Methods and Compositions for Maximizing the Prevention and/or Reduction of Various Maladies and Drug Side-Effects Using Uridine

ABSTRACT

Methods for improving the therapeutic effectiveness of uridine administered to prevent and/or reduce various maladies including adverse event from drugs are provided. Aspects of the methods include administering an effective amount of a uridine maximizing adjuvant, e.g., a 2,2′-anhydropyrimidine, or a derivative thereof, to the subject. Also provided are compositions for use in practicing the subject methods. The subject methods and compositions find use in a variety of different applications, including the treatment of a variety of different disease conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part application of InternationalApplication Serial No. PCT/US2019/046375 filed Aug. 13, 2019, whichapplication, pursuant to 35 U.S.C. § 119 (e), claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 62/718,829filed Aug. 14, 2018; the disclosures of which applications are hereinincorporated by reference.

INTRODUCTION

Multiple publications report that uridine protects in various animalmodels of human disease (Renck et al., “Human uridine phosphorylase-1inhibitors: a new approach to ameliorate 5-fluorouracil-inducedintestinal mucositis,” Invest New Drugs. (2014) 32(6):1301-7.; Jeengaret al., “Uridine Ameliorates Dextran Sulfate Sodium (DSS)-InducedColitis in Mice,” Sci Rep. (2017) 7:3924.; Chenna Narendra et al.,“Local but Not Systemic Administration of Uridine Prevents Developmentof Antigen-Induced Arthritis,” PLoS One. (2015) 10(10):e0141863.; Oh etal., “Protective effect of uridine on cornea in a rabbit dry eye model,”Invest Ophthalmol Vis Sci. (2007) 48(3):1102-9.) including, liver,(Gongalves da Silva et al., “Therapeutic effect of uridine phosphorylase1 (UPP1) inhibitor on liver fibrosis in vitro and in vivo,” Eur JPharmacol. (2020) 173670.; Le et al., Uridine prevents tamoxifen-inducedliver lipid droplet accumulation,” BMC Pharmacol Toxicol. (2014) 15:27.;Lebrecht et al., “Uridine Supplementation AntagonizesZalcitabine-induced Microvesicular Steatohepatitis in Mice,” Hepatology(2007) 45:72.) pulmonary (Orenlili Yaylagul et al., “In vivo protectiveeffect of Uridine, a pyrimidine nucleoside, on genotoxicity induced byLevodopa/Carbidopa in mice,” Food Chem Toxicol. (2015) 82:36-41.; Rozovaet al., “Uridine supplementation exerts anti-inflammatory andanti-fibrotic effects in an animal model of pulmonary fibrosis,” SciRep. (2019) 9(1):9418.; Cicko et al., “Uridine supplementation exertsanti-inflammatory and anti-fibrotic effects in an animal model ofpulmonary fibrosis,” Respir Res. (2015) 16(1):105.; Evaldsson et al.,“Anti-inflammatory effects of exogenous uridine in an animal model oflung inflammation,” International Immunopharmacology (2007) 7:1025.;Muller et al., “Local administration of uridine suppresses the cardinalfeatures of asthmatic airway inflammation,” Clinical & ExperimentalAllergy (2010) 40:1552-1560.) and neuro disease models/studies, (Cansevet al., “Neuroprotective effects of uridine in a rat model of neonatalhypoxic-ischemic encephalopathy,” Neurosci Lett. (2013) 10(542):65-70.;Goren et al., “Uridine treatment protects against neonatal brain damageand long-term cognitive deficits caused by hyperoxia,” Brain Res. (2017)1676:57-68.; Iglesias et al., “Uridine Prevents Negative Effects ofOXPHOS Xenobiotics on Dopaminergic Neuronal Differentiation,” Cells(2019) 8(11):1407.; Wang et al., “Antiepileptic effect of uridine may becaused by regulating dopamine release and receptor expression in corpusstriatum,” Brain Res. (2018) 1688:47-53.; Goren et al., “Uridinetreatment protects against neonatal brain damage and long-term cognitivedeficits caused by hyperoxia,” Brain Res. (2017) 1676:57-68.;Koyuncuoglu et al., “Uridine protects against hypoxic-ischemic braininjury by reducing histone deacetylase activity in neonatal rats,”Restor Neurol Neurosci. (2015) 33(5):777-84.; Liu et al., “Uridinedecreases morphine-induced behavioral sensitization by decreasing dorsalstriatal dopamine release possibly via agonistic effects at GABAreceptors,” European Neuropsychopharmacology (2014) 24(9):1557-66.;Saydoff et al., “Uridine prodrug improves memory in Tg2576 and TAPP miceand reduces pathological factors associated with Alzheimer's disease inrelated models,” J Alzheimers Dis. (2013) 36(4):637-57.; Dobolyi et al.,“Uridine function in the central nervous system,” Curr Top Med Chem.(2011) 11(8):1058-67.; Amante et al., “Uridine ameliorates thepathological phenotype in transgenic G93A-ALS mice,” Amyotroph LateralScler. (2010) 11(6):520-30.; Cansev et al., “Restorative effects ofuridine plus docosahexaenoic acid in a rat model of Parkinson'sdisease,” Neurosci Res. (2008) 62(3):206-9.; Saydoff et al., “Oraluridine pro-drug PN401 is neuroprotective in the R6/2 and N171-82Q mousemodels of Huntington's disease,” Neurobiol Dis. (2006) 24(3):455-65.;Venhoff et al., “Oral uridine supplementation antagonizes the peripheralneuropathy and encephalopathy induced by antiretroviral nucleosideanalogues,” AIDS. (2010) 24(3):345-52.; Cansev et al., “Neuroprotectiveeffects of uridine in a rat model of neonatal hypoxic-ischemicencephalopathy,” Neurosci Lett. (2013) 542:65-70.) as well asmodels/studies reflecting mitochondrial dysfunction. (Lebrecht et al.,“Uridine Supplementation Antagonizes Zidovudine-induced mitochondrialmyopathy & hyperlactatemia in mice,” Arthritis Rheum (2008) 58:318-326.;Mironova et al., “Dynamic Restructuring of the Myocardial Mitochondriain Response to Uridine Modulation of the Activity of MitochondrialATP-Dependent Potassium Channel under Conditions of Acute HypoxicHypoxia,” Bull Exp Biol Med. (2019) 166(6):806-810.; Balcarek et al.,“Role of pyrimidine depletion in the mitochondrial cardiotoxicity ofnucleoside analogue reverse transcriptase inhibitors,” J Acquir ImmuneDefic Syndr. (2010) 55(5):550-7.).

One example of the use of uridine to reduce a drug-induced side effectsis reduction of mucositis post treatment with 5-fluorouracil (5-FU), afluorinated analogue of uracil. 5-FU is sold under the trade namesAdrucil, Carac, Efudex and Efudix among others. It is one of the oldesttumor antimetabolites, and one of the most effective. It was developedin the 1950's and since then has been employed for the treatment ofvarious solid tumors, such as prostate, pancreatic, colon, rectum,breast, liver, head, neck and bladder carcinomas. In the cell, 5-FU isconverted to the active nucleotides 5-fluoro-2′-deoxyuridine5′-monophosphate (5-FdUMP), 5-fluorouridine 5′-triphosphate (5-FUTP) and5-fluoro-2′-deoxyuridine 5′-triphosphate (5-FdUTP). The metabolism isvery complex. 5-FU is first converted by phosphoribosyl transferase to5-fluorouridine 5′-monophosphate (5-FUMP), which is phosphorylated bymeans of nucleotide kinases through the 5′-diphosphate (5-FUDP) to formthe 5′-triphosphate (5-FUTP). The nucleotide 5-FUTP is incorporated byRNA polymerases into the RNA instead of UTP and thus interferes with thefunction of the RNA. The metabolite 5-FUDP is converted with the aid ofribonucleotide reductase to 5-FdUDP, which is then phosphorylated bynucleoside diphosphate kinase to form 5-FdUTP. 5-FdUTP may also beincorporated into DNA as a false building block by DNA polymerases.Removal of the wrong nucleotides by uracil glycosylase results in DNAsingle strand breaks, which leads to inhibition of DNA synthesis, DNAfragmentation and eventually apoptosis. Dephosphorylation of 5-FdUTP bymeans of dUTPase forms the third active metabolite 5-fluorodeoxyuridinemonophosphate (5-FdUMP). It inhibits thymidylate synthase (TS), whichcatalyzes the reductive methylation of deoxyuridine monophosphate (dUMP)to deoxythymidine monophosphate (dTMP) together with the cofactor5,10-methylenetetrahydrofolate. After binding of 5-FdUMP to TS, theenzyme is blocked in a tertiary complex (TS, 5-FdUMP and folate),whereby methylation at the C-5 is inhibited. This results in inhibitionof DNA synthesis.

Uridine is reported to minimize the myelosuppression associated with5-FU treatment. (van Groeningen et al., “Reversal of5-fluorouracil-induced toxicity by oral administration of uridine,” AnnOncol. (1993) 4(4):317-20.; van Groeningen et al., “Reversal of5-fluorouracil-induced myelosuppression by prolonged administration ofhigh-dose uridine,” J Natl Cancer Inst. (1989) 18; 81(2):157-62.) Severemucositis is associated 5-FU treatment of proliferative diseases such asa cancer, particularly colon cancer. (Baydar et al., “Prevention of oralmucositis due to 5-fluorouracil treatment with oral cryotherapy,” J NatlMed Assoc (2005) 97(8):1161-4.). Studies have also suggested thaturidine administration can minimize the mucositis associated with 5-FUtreatment (Bagrij et al., “Influence of uridine treatment in mice on theprotection of gastrointestinal toxicity caused by 5-fluorouracil,”Anticancer Res. (1993) 13(3):789-93.; Kralovanszky et al., “Biochemicalconsequences of 5-fluorouracil gastrointestinal toxicity in rats; effectof high-dose uridine,” Cancer Chemother Pharmacol. (1993) 32(3):243-8.).To avoid any possibility of interfering with the efficacy of 5-FU, theuridine must be administered several hours after the 5-FU.

Mucositis is a profoundly serious, often treatment limiting, adverseevent that often develops as a complication of chemotherapy, including5-FU therapy. Mucositis is characterized by swelling, irritation, anddiscomfort of mucosal linings such as those of the gastrointestinal (GI)tract, including oral and oralpharyngeal cavities, as well as nasal,optical, vaginal and rectal mucosa. Mucositis can result in mouth andthroat sores, diarrhea, abdominal cramping and tenderness, and rectalulcerations.

As an inflammation of mucous membranes which often involves infectionand/or ulceration, mucositis is a serious and often very painfuldisorder. Exposure to radiation often results in significant disruptionof cellular integrity in mucosal epithelium and the underlyingconnective tissue, leading to inflammation, infection and/or ulcerationat mucosal sites such as, for example, in the mouth, throat and otherportions of the GI tract.

Mucositis adversely impacts the quality of life of cancer patients inseveral ways. Patients may experience intense pain, nausea andgastro-enteritis. The mouth and throat sores of mucositis can causesignificant pain and make it difficult to eat, drink, and even take oralmedication. In general, symptoms of mucositis appear within five to tendays after the start of cancer treatment and can last several weeksafter cessation of treatment.

The incidence of mucositis, as well as its severity, depends on factorssuch as the type and duration of the cancer treatment. Mucositis affects76-100 percent of patients receiving higher doses of chemotherapy forbone marrow transplantation. Mucositis afflicts over 400,000 patients ayear in the US, and the incidence is growing as the need for radiationand chemotherapy treatments grows. The severity of mucositis can limitsubsequent doses of chemotherapy.

Efforts to counter the discomforts of mucositis can lead to disruptionsin cancer treatment, alterations in treatment dosages, or shifting todifferent modes of treatment. Severe mucositis can also lead to the needfor parenteral nutrition or hospitalization for intravenous feedingbecause of the mouth ulcers, cramps, extreme pain, gut denuding, andsevere diarrhea. Thus, the development of effective approaches topreventing and treating mucositis is important for improving the care ofcancer patients.

Mucositis patients are highly susceptible to infection, as a breach inthe otherwise protective linings of the oral mucosa and gastrointestinaltract can have serious consequences. The GI tract is colonized by a vastarray of microorganisms, and mucosal lesions can serve as portals ofentry for endogenous microorganisms, becoming sites of secondaryinfection.

As previously mentioned, and referenced, uridine shows promise as atherapeutic for various conditions, including chemotherapy-inducedmucositis. However, direct administration of uridine is not a realisticas a drug because of its extremely short elimination half-life (t½).(Deng et al., “An adipo-biliary-uridine axis that regulates energyhomeostasis,” Science (2017) 355(6330): eaaf5375.). Uridine isessentially cleared in a single pass of blood through the liver,primarily by uridine phosphorylase (UPase), which is replaced in ahighly regulated manner by new uridine formed by de novo synthesis.(Gasser et al., “Novel single pass exchange of circulating uridine inrat liver,” Science (1981) 213:777-778.). In addition, po administrationof uridine is associated with considerable GI dysfunction. For example,clinical studies of high-dose oral uridine have been unsuccessful due tosevere patient diarrhea, phlebitis, and pyrogenic reactions. Continuousinfusions of high-dose uridine have also been restricted because oftoxicities, including high fever, cellulitis, and superior vena cavasyndrome. (van Groeningen et al., “Clinical and pharmacologic study oforally administered uridine,” J Natl Cancer Inst. (1991) 83:437-41.; vanGroeningen et al., “Modulation of fluorouracil toxicity with uridine,”Semin Oncol. (1992) 9(2 Suppl 3):48-54.). Approaches have been tried toovercome the limitations of uridine administration. For example,prodrugs have been developed and tested, e.g., tri-acetyl uridine whichhas been approved for marketing to treat inter alia 5-FU overdose. (Maet al., “Emergency use of uridine triacetate for the prevention andtreatment of life-threatening 5-fluorouracil and capecitabine toxicity,”Cancer. (2017) 123(2):345-356.).

SUMMARY

Methods for maximizing the therapeutic effect of treatment with uridineor a uridine prodrug are provided. One example provided is moreeffectively reducing 5-FU-induced mucositis in a subject. Aspects ofthis method include administering to the subject an effective amount ofuridine or a uridine prodrug to reduce toxicity, e.g., mucositis, fromthe 5-FU treatment along with an agent to maximize the effectiveness ofthe administered uridine or administered uridine prodrug. In certainembodiments, the maximizing agent is a 2,2′-anhydropyrimidine, or aderivative thereof. Also provided are compositions for use in practicingthe subject methods. The subject methods and compositions find use in avariety of different applications, including the treatment of a varietyof different disease conditions, e.g., that would benefit from thetherapeutic effectiveness of a uridine or uridine prodrug treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the chemical structures of 5-fluorouracil (5-FU),TK-112690 (TK-90), and uridine.

FIG. 2 depicts percent change in CD-1 female mouse weight for Day 5(termination) versus Day 0. Animals were dosed with either PBS, ip, for5 days; 80 mg/kg 5-FU, bid, ip, for 5 days; or 80 mg/kg 5-FU, bid, ip;and 120 mg/kg TK-90, bid, for 5 days. Six mice were used for alltreatment groups. In this study, uridine+TK-90 protected from the weightloss induced by the 5-FU better than uridine alone.

FIG. 3 depicts percent change in CD-1 male mouse weight for Day 5(termination) versus Day 0. Animals were dosed with either PBS ip for 5days; 400 mg/kg 5-FU, bid for one day, ip; or 400 mg/kg 5-FU, ip, bidfor one day plus 500 mg/kg, bid, ip, uridine for three days; or 400mg/kg 5-FU, ip, bid for one day plus 500 mg/kg, bid, ip, uridine forthree days and 100 mg/kg TK-90, ip, qd for three days. Four mice wereused for the PBS group all other groups were eight mice. Like the studysummarized in FIG. 2, uridine+TK-90 protected from the weight lossinduced by the 5-FU better than uridine alone.

FIG. 4 depicts citrulline concentrations measured in plasma obtained atsacrifice on Day 3 from C57BL/6 female mice treated on Day 0 with 200mg/kg 5-FU, ip. On Days 0-2, the uridine and uridine+TK-90 group animalswere dosed with 250 mg uridine, bid. On Days 0-2, the uridine+TK-90group animals were treated with 700 mg/kg TK-90, bid, in addition to the5-FU on Day 0 and the uridine on Days 0-2. Animals in the PBS group weretreated on Day 0-2 with PBS, bid. Data from all six mice in the PBS,uridine and uridine plus TK-90 groups were considered. Data from onlyfive animals in the 5-FU group were considered because results from oneanimal was considered an outlier (unusually low response from theinjected 5-FU). Plasma citrulline is a marker for mucosal health. (Joneset al., “Citrulline as a Biomarker in the Murine Total-Body IrradiationModel: Correlation of Circulating and Tissue Citrulline to SmallIntestine Epithelial Histopathology,” Health Phys. (2015) 109(5):452-65.). Plasma citrulline is lowered with mucositis. (Fragkos et al.,“Citrulline as a marker of intestinal function and absorption inclinical settings: A systematic review and meta-analysis,” UnitedEuropean Gastroenterol J. (2018) 6(2):181-191.). TK-90 plus uridinereverses the decrease in plasma citrulline caused by the 5-FU betterthan uridine alone.

DEFINITIONS

When describing the compounds, pharmaceutical compositions containingsuch compounds, and methods of using such compounds and compositions,the following terms have the following meanings unless otherwiseindicated. It should also be understood that any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions are intended to include such substitutedmoieties within their scope.

“Acyl” refers to a radical —C(O)R, where R is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroalkyl, orheteroaryl as defined herein. Representative examples include, but arenot limited to, formyl, acetyl, cylcohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

“Acylamino” refers to a radical —NR′C(O)R, where R′ is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl and R is hydrogen, alkyl, alkoxy, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl orheteroarylalkyl, as defined herein. Representative examples include, butare not limited to, formylamino, acetylamino, cyclohexylcarbonylamino,cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino andthe like.

“Acyloxy” refers to the group —OC(O)H, —OC(O)-alkyl, —OC(O)-aryl or—OC(O)-cycloalkyl.

“Aliphatic” refers to hydrocarbyl organic compounds or groupscharacterized by a straight, branched or cyclic arrangement of theconstituent carbon atoms and an absence of aromatic unsaturation.Aliphatics include, without limitation, alkyl, alkylene, alkenyl,alkynyl and alkynylene. Aliphatic groups typically have from 1 or 2 to 6or 12 carbon atoms.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbylgroups having up to about 11 carbon atoms, particularly, from 2 to 8carbon atoms, and more particularly, from 2 to 6 carbon atoms, which canbe straight-chained or branched and having at least 1 and particularlyfrom 1 to 2 sites of olefinic unsaturation. Particular alkenyl groupsinclude ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl(—C(CH₃)═CH₂), vinyl and substituted vinyl, and the like.

“Alkoxy” refers to the group —O-alkyl. Particular alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, andthe like.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Alkoxycarbonylamino” refers to the group —NRC(O)OR′ where R ishydrogen, alkyl, aryl or cycloalkyl, and R′ is alkyl or cycloalkyl.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 12 or 18 carbon atoms, more particularlyas a lower alkyl, from 1 to 8 carbon atoms and still more particularly,from 1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, n-octyl, tert-octyl and the like. The term “alkyl” alsoincludes “cycloalkyls” as defined herein.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 12 or 18 carbon atoms and moreparticularly 1 to 6 carbon atoms which can be straight-chained orbranched. This term is exemplified by groups such as methylene (—CH₂—),ethylene (—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂— and—CH(CH₃)CH₂—) and the like.

“Alkynyl” refers to acetylenically unsaturated hydrocarbyl groupsparticularly having up to about 12 or 18 carbon atoms and moreparticularly 2 to 6 carbon atoms which can be straight-chained orbranched and having at least 1 and particularly from 1 to 2 sites ofalkynyl unsaturation. Particular non-limiting examples of alkynyl groupsinclude acetylenic, ethynyl (—C≡CH), propargyl (—CH₂C≡CH), and the like.

“Amino” refers to the radical —NH2.

“Amino acid” refers to any of the naturally occurring amino acids (e.g.,Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D, L, or DL form. Theside chains of naturally occurring amino acids are well known in the artand include, for example, hydrogen (e.g., as in glycine), alkyl (e.g.,as in alanine, valine, leucine, isoleucine, proline), substituted alkyl(e.g., as in threonine, serine, methionine, cysteine, aspartic acid,asparagine, glutamic acid, glutamine, arginine, and lysine), alkaryl(e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g.,as in tyrosine), and heteroarylalkyl (e.g., as in histidine).

“Aminocarbonyl” refers to the group —C(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalkyl, or where the R groupsare joined to form an alkylene group.

“Aminocarbonylamino” refers to the group —NRC(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalkyl, or where two R groupsare joined to form an alkylene group.

“Aminocarbonyloxy” refers to the group —OC(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalky, or where the R groupsare joined to form an alkylene group.

“Amino-containing saccharide group” refers to a saccharide group havingan amino substituent. Representative amino-containing saccharide includeL-vancosamine, 3-desmethyl-vancosamine, 3-epi-vancosamine,4-epi-vancosamine, acosamine, actinosamine, daunosamine,3-epi-daunosamine, ristosamine, N-methyl-D-glucamine and the like.

“Aralkyl” or “arylalkyl” refers to an alkyl group, as defined above,substituted with one or more aryl groups, as defined above.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like. Particularly, anaryl group comprises from 6 to 14 carbon atoms.

“Aryloxy” refers to —O-aryl groups wherein “aryl” is as defined herein.

“Autoimmune disease” or “autoimmune condition” refers an illness thatoccurs when the body tissues are attacked by its own immune system.Examples of autoimmune disease or conditions include multiple sclerosis,ankylosing spondylitis, Crohn's disease, arthritis, psoriasis, Behçet'sdisease and psoriatic arthritis.

Azido” refers to the radical —N₃.

Boxplots refers to graphical rendition of statistical data based on theminimum, first quartile, median, third quartile, and maximum. The term“box plot” comes from the fact that the graph looks like a rectanglewith lines extending from the top and bottom. In a typical box plot, thetop of the rectangle indicates the third quartile, a horizontal linenear the middle of the rectangle indicates the median, and the bottom ofthe rectangle indicates the first quartile. A vertical line extends fromthe top of the rectangle to indicate the maximum value, and anothervertical line extends from the bottom of the rectangle to indicate theminimum value.

“Carbohydrate” means a mono-, di-, tri-, or polysaccharide, wherein thepolysaccharide can have a molecular weight of up to about 20,000, forexample, hydroxypropyl-methylcellulose or chitosan. “Carbohydrate” alsoencompasses oxidized, reduced or substituted saccharide monoradicalcovalently attached to the anhydropyrimidine (e.g., anhydrothymidine oranhydrouridine), or derivative thereof any atom of the saccharidemoiety, e.g., via the aglycone carbon atom. The “mono-, di-, tri-, orpolysaccharide” can also include amino-containing saccharide groups.Representative “carbohydrate” include, by way of illustration, hexosessuch as D-glucose, D-mannose, D-xylose, D-galactose, vancosamine,3-desmethyl-vancosamine, 3-epi-vancosamine, 4-epi-vancosamine,acosamine, actinosamine, daunosamine, 3-epi-daunosamine, ristosamine,D-glucamine, N-methyl-D-glucamine, D-glucuronic acid,N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, sialyic acid, iduronicacid, L-fucose, and the like; pentoses such as D-ribose or D-arabinose;ketoses such as D-ribulose or D-fructose; disaccharides such as2-O-(α-L-vancosaminyl)-β-D-glucopyranose-,2-O-(β-desmethyl-α-L-vancosaminyl)-β-D-glucopyranose, sucrose, lactose,or maltose; derivatives such as acetals, amines, acylated, sulfated andphosphorylated sugars; oligosaccharides having from 2 to 10 saccharideunits. The saccharides can be either in their open, r pyranose orfuranose forms.

“Carboxyl” refers to the radical —C(O)OH.

“Cyano” refers to the radical —CN.

“Cycloalkenyl” refers to cyclic hydrocarbyl groups having from 3 to 10carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems and having at least oneand particularly from 1 to 2 sites of olefinic unsaturation. Suchcycloalkenyl groups include, by way of example, single ring structuressuch as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.

“Cycloalkyl” refers to cyclic hydrocarbyl groups having from 3 to about10 carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems, which optionally can besubstituted with from 1 to 3 alkyl groups. Such cycloalkyl groupsinclude, by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,2-methylcyclopentyl, 2-methylcyclooctyl, and the like, and multiple ringstructures such as adamantanyl, and the like.

“Heterocycloalkyl” refers to a stable heterocyclic non-aromatic ring andfused rings containing one or more heteroatoms independently selectedfrom N, O and S. A fused heterocyclic ring system may includecarbocyclic rings and need only include one heterocyclic ring. Examplesof heterocyclic rings include, but are not limited to, piperazinyl,homopiperazinyl, piperidinyl and morpholinyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo. Halogroups can be either fluoro or chloro.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocycloalkyl, aryl,e.g., heteroaryl, cycloalkenyl, e.g., heterocycloalkenyl,cycloheteroalkenyl, e.g., heterocycloheteroalkenyl and the like havingfrom 1 to 5, and particularly from 1 to 3 heteroatoms. A heteroatom isany atom other than carbon or hydrogen and is typically, but notexclusively, nitrogen, oxygen, sulfur, phosphorus, boron, chlorine,bromine, or iodine. An unsubstituted heteroatom refers to a pendantheteroatom such as an amine, hydroxyl and thiol. A substitutedheteroatom refers to a heteroatom that is other than a pendantheteroatom.

“Heteroaryl” refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. The heteroaryl group can bea 5-20 membered heteroaryl, or 5-10 membered heteroaryl. Particularheteroaryl groups are those derived from thiophen, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole and pyrazine.

“Hydroxyl” refers to the radical —OH.

“Nitro” refers to the radical —NO₂.

“Peptide” refers to a polyamino acid containing up to 2, 5, 10, or about100 amino acid residues.

“Polypeptide” means polyamino acid containing from about 100 amino acidunits to about 1,000 amino acid units, from about 100 amino acid unitsto about 750 amino acid units, or from about 100 amino acid units toabout 500 amino acid units.

“Proliferative disease” or “proliferative condition” refers to a diseaseor condition featuring pathologic growth as an underlying pathology.Examples include cancer, arthritis and psoriasis.

“Side-effect” means an undesirable adverse consequence of drugadministration such as mucositis associated with administration of 5-FU.

“Stereoisomer” as it relates to a given compound is well understood inthe art, and refers to another compound having the same molecularformula, wherein the atoms making up the other compound differ in theway they are oriented in space, but wherein the atoms in the othercompound are like the atoms in the given compound with respect to whichatoms are joined to which other atoms (e.g., an enantiomer, adiastereomer, or a geometric isomer). See for example, Morrison andBoyd, Organic Chemistry, 1983, 4th ed., Allyn and Bacon, Inc., Boston,Mass., p. 123.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).“Substituted” groups particularly refer to groups having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, selected from the group consisting of acyl,acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl,alkoxycarbonylamino, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, aralkyl, azido,carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl,imidate, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkylthio, (substituted alkyl)thio, arylthio,(substituted aryl)thio, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— andaryl-S(O)₂. Typical substituents include, but are not limited to, —X,—R⁸ (with the proviso that R⁸ is not hydrogen), —O—, ═O, —OR⁸, —SR⁸,—S⁻, ═S, —NR⁸R⁹, ═NR⁸, —CX₃, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(O)₂O⁻, —S(O)₂OH, —S(O)₂R⁸, —OS(O₂)O⁻, —OS(O)₂R⁸, —P(O)(O—)₂,—P(O)(OR⁸)(O⁻), —OP(O)(OR⁸)(OR⁹), —C(O)R⁸, —C(S)R⁸, —C(O)OR⁸,—C(O)NR⁸R⁹, —C(O)O⁻, —C(S)OR⁸, —NR¹⁰C(O)NR⁸R⁹, —NR¹⁰C(S)NR⁸R⁹,—NR¹¹C(NR¹⁰)NR⁸R⁹ and —C(NR¹⁰)NR⁸R⁹, where each X is independently ahalogen.

“Substituted amino” includes those groups recited in the definition of“substituted” herein, and particularly refers to the group —N(R)₂ whereeach R is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, and whereboth R groups are joined to form an alkylene group.

“Thioalkoxy” refers to the group —S-alkyl.

“Thioaryloxy” refers to the group —S-aryl.

“Thioketo” refers to the group ═S.

“Thiol” refers to the group —SH.

TK-112690 (TK-90), a 2,2′-anhydropyrimidine, is an authentic UPaseinhibitor (Cao et al., “Uridine phosophorylase: an important enzyme inpyrimidine metabolism and fluoropyrimidine activation,” Drugs Today(Barc). (2004) 40:431-4) that has successfully completed its thirdclinical trial as a mitigator of chemotherapy-induced mucositis. TK-90has no known side effects, has proven efficacy as an agent to mitigatechemotherapy-induced mucositis, is readily synthesized, and is activewhen dosed parenterally or orally. A UPase inhibitor, structurallyvastly different from TK-90 is reported to protect from 5-flourouracilinduced mucositis. (Renck et al., “Human uridine phosphorylase-1inhibitors: a new approach to ameliorate 5-fluorouracil-inducedintestinal mucositis,” Invest New Drugs (2014) 32(6):1301-7.).Inhibition of uridine phosphorylase causes elevated uridine. Uridine isan endogenous biochemical which is known to protect in multiple animalmodels of human disease (Renck et al., “Human uridine phosphorylase-1inhibitors: a new approach to ameliorate 5-fluorouracil-inducedintestinal mucositis,” Invest New Drugs. (2014) 32:1301-7; Jeengar etal., “Uridine Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitisin Mice,” Sci Rep. (2017) 7:3924; Chenna et al., “Local but Not SystemicAdministration of Uridine Prevents Development of Antigen-InducedArthritis,” PLoS One. (2015)₁₀: e0141863; Oh et al., “Protective effectof uridine on cornea in a rabbit dry eye model,” Invest Ophthalmol VisSci. (2007) 48:1102-9; Gonçalves da Silva et al., “Therapeutic effect ofuridine phosphorylase 1 (UPP1) inhibitor on liver fibrosis in vitro andin vivo,” Eur J Pharmacol. (2020) 22 Oct. 2020:173670; Rozova et al.,“Uridine supplementation exerts anti-inflammatory and anti-fibroticeffects in an animal model of pulmonary fibrosis,” Sci Rep. (2019) 9:9418; Cicko et al., “Uridine supplementation exerts anti-inflammatoryand anti-fibrotic effects in an animal model of pulmonary fibrosis,”Respir Res. (2015) 16:105; Cansev et al., “Neuroprotective effects ofuridine in a rat model of neonatal hypoxic-ischemic encephalopathy,”Neurosci Lett. (2013) 542: 65-70; Goren et al., “Uridine treatmentprotects against neonatal brain damage and long-term cognitive deficitscaused by hyperoxia,” Brain Res. (2017) 1676: 57-68; Wang et al.,“Uridine treatment protects against neonatal brain damage and long-termcognitive deficits caused by hyperoxia,” Brain Res. (2018) 1688:47-53;Venhoff et al., “Oral uridine supplementation antagonizes the peripheralneuropathy and encephalopathy induced by antiretroviral nucleosideanalogues,” AIDS. (2010) 24(3):345-52; Cansev et al., “Neuroprotectiveeffects of uridine in a rat model of neonatal hypoxic-ischemicencephalopathy,” Neurosci Lett. (2013) 542:65-70).

“Uridine phosphorylase” refers in enzymology to a phosphorylase (EC2.4.2.3) that catalyzes the chemical reaction:uridine+phosphate→uracil+alpha-D-ribose 1-phosphate. The two substratesof this enzyme are uridine and phosphate, whereas its two products areuracil and alpha-D-ribose 1-phosphate. This enzyme belongs to the familyof glycosyltransferases, specifically the pentosyltransferases. Thesystematic name of this enzyme class is uridine: phosphatealpha-D-ribosyltransferase. Other names in common use include pyrimidinephosphorylase, UrdPase, UPH, and UPase. This enzyme participates inpyrimidine metabolism. The clearance of uridine is controlled by UPase,and the inhibition of UPase leads to an increase in uridine (⬆ uridinesalvage). (Pizzorno et al., “Homeostatic control of uridine and the roleof uridine phosphorylase: a biological and clinical update,” BiochimBiophys Acta. (2002) 1587(2-3):133-44.). This provides a target for drugdiscovery and development.

“Uridine Supplement” refers to either a formulated product containinguridine or a formulated product containing a uridine precursor such asuridine monophosphate or acetylated uridine that converts to uridine inthe body. The formulated product could be a solution, a capsule, atablet or a cream. The product could be administered po, ip, sc, or iv.The uridine supplement could be administered as part of a more complexmixture such as a nutritional supplement.

Miscellaneous definitions are as follows. ip, po and sc refer tointraperitoneal, oral or subcutaneous dosing, respectively. SD isstandard deviation and SE is standard error. PBS is phosphate bufferedsaline. q.d. and b.i.d refer to once a day and twice-a-day,respectfully.

One having ordinary skill in the art will recognize that the maximumnumber of heteroatoms in a stable, chemically feasible heterocyclicring, whether it is aromatic or non-aromatic, is determined by the sizeof the ring, the degree of unsaturation and the valence of theheteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

DETAILED DESCRIPTION

Numerous reports in the scientific literature suggest that uridine oruridine prodrugs can be used to treat or prevent various maladiesincluding drug-induced adverse events (see conditions listed above).Aspects of the invention are intended to be used with uridine to improvethe efficacy of a uridine treatment by decreasing the clearance ofuridine and thereby overcoming the unusually short half live of thenucleoside. The decreased clearance is achieved by inhibiting the enzymeprincipally responsible for metabolizing uridine, UPase.

An embodiment of the invention is provided by improving the knownability of uridine to suppress mucositis produced by a commonly usedchemotherapeutic 5-FU. As such, methods for reducing 5-FU inducedtoxicity, e.g., mucositis, in a subject are provided. Aspects of themethods include administering an effective amount of a uridine and auridine maximizing adjuvant to the subject. In certain embodiments, theuridine maximizing adjuvant (which when employed to ameliorate F-FUinduced toxicity may be referred to as a fluorouracil or F-Fu toxicityreducing adjuvant, is a 2,2′-anhydropyrimidine, or a derivative thereof.Also provided are compositions for use in practicing the subjectmethods. The subject methods and compositions find use in a variety ofdifferent applications, including the treatment of a variety ofdifferent disease conditions.

Of particular interest is the use of anhydro-nucleosides as adjuvants toameliorate the toxic side-effects of 5-FU therapy, as well ascompositions for practicing the subject methods and other applications.Anhydro-nucleosides are analogs of natural nucleosides, often findinguse as intermediates in the synthesis of nucleoside derivatives. Theyare characterized by having, in addition to the N-glycoside linkage, acovalent linkage either directly or via bridging atoms between the 2′,3′, or 5′ carbons of the sugar and a carbon, oxygen or nitrogen atom(other than the nitrogen of the glycoside bond) of the base. Theanhydro-pyrimidines are characterized by a pyrimidine base that iscovalently linked either directly or via bridging atoms between the 2′,3′, or 5′ carbons of the sugar and a carbon, oxygen or nitrogen atom(other than the nitrogen of the glycoside bond) of the pyrimidine base.The uridine maximizing adjuvant 2,2′-anhydropyrimidine and derivativesthereof are of specific interest.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

While the method has or will be described for the sake of grammaticalfluidity with functional explanations, it is to be expressly understoodthat the claims, unless expressly formulated under 35 U.S.C. § 112, arenot to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

In further describing the subject invention, the subject methods aredescribed first in greater detail, followed by a review of the variouscompositions, e.g., formulations and kits, that may find use in thesubject methods, as well as a discussion of various representativeapplications in which the subject methods and compositions find use.

Methods

As summarized above, the subject invention provides methods of enhancinguridine activity, e.g., to prevent or treat a condition for whichuridine therapy is helpful. Any condition in which uridine therapyprovides a benefit may be treated in accordance with the presentinvention. Conditions of interest may vary, and include, but are notlimited to: chemotherapy resultant conditions, e.g., mucositis, causedby chemotherapy, e.g., 5-FU therapy; lipodystrophy, e.g., associatedwith the usage of nucleoside reverse transcriptase inhibitors for HIVtreatment; hepatic steatosis e.g., induced by zalcitabine, fenofibrate,and tamoxifen; orotic aciduria; Colitis, arthritis, corneal conditions,liver conditions, pulmonary conditions, neurological conditions,mitochondrial conditions; and the like.

In some instances, the condition that is prevented or treated ismucositis associated with 5-FU therapy. As such, embodiments of theinvention include administering a 5-FU therapy to a subject in needthereof, e.g., for the treatment of a host suffering from disease orcondition treatable by a 5-FU therapy (as described in greater detailbelow). An aspect of the subject methods is that the 5-FU therapy isadministered to the subject in combination with a uridine maximizingadjuvant. In certain embodiments, the uridine maximizing adjuvant is a2,2′-anhydropyrimidine, such as a 2,2′-anhydrouridine oranalogue/derivative thereof. By “in combination with”, is meant that anamount of the uridine maximizing adjuvant is administered anywhere fromsimultaneously to up to 5 hours or more, e.g., 10 hours, 15 hours, 20hours or more, prior to, or after, the 5-FU therapy. In certainembodiments, the 5-FU therapy and uridine maximizing adjuvant areadministered sequentially, e.g., where the 5-FU is administered beforeor after the uridine maximizing adjuvant. In yet other embodiments, the5-FU therapy and uridine maximizing adjuvant are administeredsimultaneously, e.g., where the 5-FU therapy and uridine maximizingadjuvant are administered at the same time as two separate formulations,or are combined into a single composition, that is administered to thesubject. Regardless of whether the 5-FU therapy and uridine maximizingadjuvant are administered sequentially or simultaneously, as illustratedabove, or any effective variation thereof, the agents are considered tobe administered together or in combination for purposes of the presentinvention. Routes of administration of the two agents may vary, whererepresentative routes of administration are described in greater detailbelow.

In the subject methods, an effective amount of a 5-FU therapy activeagent is administered to a host in need thereof in combination with aneffective amount of a uridine maximizing adjuvant. By “5-FU therapy” ismeant administration of one or more 5-FU active agents. By “5-FU activeagent” is meant 5-fluoro-2,4(1H,3H)-Pyrimidinedione (i.e., 5-FU) or ananalogue/derivative thereof. In some instances, a given 5-FU therapy mayemploy 5-FU or a derivative thereof, which may be present in a varietyof different formulations. 5-FU therapies that may be employed inembodiments of the invention include, but are not limited to, thosedescribed in U.S. Pat. Nos. 9,902,752; 9,012,444; 8,889,699; 8,492,413;8,440,398; 8,101,652; 7,910,580; 7,622,458; 7,345,039; 7,026,301;6,794,370; 6,670,335; 6,403,569; 5,843,917; 5,676,973; 5,663,321;5,610,160; 5,496,810; 5,457,187; 5,116,600; 5,089,503; 5,077,055;5,049,551; 5,047,521; 4,983,609; 4,914,105; 4,864,021; 4,810,790;4,719,213; 4,704,393; 4,652,570; 4,650,801; 4,631,342; 4,622,325;4,605,738; 4,558,12; 4,507,301; 4,497,815; 4,481,203; 4,408,048;4,394,505; 4,371,535; 4,349,552; 4,336,381; 4,328,229; 4,256,885;4,249,006; 4,248,999; 4,206,208; 4,196,202; 4,186,266; 4,169,201;4,130,648; 4,124,765; 4,122,251; 4,121,037; 4,113,949; 4,110,537;4,107,162; 4,088,646; 4,071,519; 4,032,524; 4,029,661; 3,971,784;3,960,864; 3,954,759; and 3,948,897; the disclosures of which are hereinincorporated by reference.

5-FU active agents of the present invention include5-fluoro-2,4(1H,3H)-Pyrimidinedione and any analogues/derivativesthereof whose toxicity is reduced when administered in conjunction witha toxicity-reducing adjuvant according to the subject invention. Whetheror not a given 5-FU active agent is suitable for use according to thepresent invention can be readily determined using assays employed in theexperimental section, below. In certain embodiments, the 5-FU activeagent is one whose occurrence and/or intensity of observable toxicside-effects are reduced by the uridine maximizing adjuvant as observedin the mouse assay described in the Experimental section below, e.g., by2-fold or more, such as 5-fold or more, including 10-fold or more.

The phrase “uridine maximizing adjuvant” refers to an agent that reducesthe clearance of uridine. Uridine maximizing adjuvants of interest arethose agents that reduce the occurrence and/or intensity of observabletoxic side-effects of a given 5-FU active agent, as observed in themouse assay described in the Experimental section below, e.g., by 2-foldor more, such as 5-fold or more, including 10-fold or more. Aspects oftoxicity-reducing adjuvants according to certain embodiments of theinvention are that the adjuvants do not substantially reduce, and incertain embodiments have no impact at all, on the cytotoxicity of the5-FU active agent.

The uridine maximizing adjuvants of interest are 2,2′-anhydropyrimidinesand derivatives thereof. In some embodiments, the 2,2′-anhydropyrimidineor derivative thereof is a compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof.

wherein:

each R¹, R², R³ and R⁴ is independently selected from the groupconsisting of hydrogen, substituted or unsubstituted heteroatom,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted aralkyl, hydroxyl, halogen, azido, amino,substituted amino, carbohydrate, nucleic acid, amino acid, peptide, dye,fluorophore, and polypeptide.

In certain embodiments, the compound is of formula (I), R¹, R², R³ andR⁴ are independently hydrogen, hydroxyl, heteroatom, C₁-C₁₈ alkyl,C₁-C₁₈ substituted alkyl, C₁-C₁₈ alkenyl, C₁-C₁₈ acyl, amino,substituted amino, wherein the alkyl, alkenyl or acyl is linear orbranched, and optionally substituted with a hydroxyl, an ester and itsderivatives, a carboxyl and its derivatives, a cycloalkyl, aheterocycloalkyl, an aryl, a heteroaryl, an aralkyl, a heteroatom, andpossibly containing in chain or bridging heteroatoms such as nitrogen,oxygen, and sulfur.

Examples of R¹ constituents of interest include, but are not limited to:hydrogen; hydroxyl; sulfyhydryl; halogen such as fluorine, chlorine,bromine or iodine, as well as pseudohalogen such as a loweralkylsulfonyl group of 1 to 5 carbons such as methyl-, ethyl-, propyl-,isopropyl-, butyl-, isobutyl-, tert-butyl-, and pentasulfonyl orarylsulfonyl such as benzene, p-toluene, p-nitrobenzenesulfonyl groups;lower alkyl containing 1 to 20 carbons such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl and the like, includingsubstituted lower alkyl such as aminomethyl, hydroxymethyl, methoxy,ethyloxy, propyloxy, benzyloxy, imidate, alkylthio, (substitutedalkyl)thio, arylthio, (substituted aryl)thio and the like; lower alkenylcontaining 1 to 20 carbons such as vinyl and substituted vinyl, ethynyland substituted ethynyl, where the substituted vinyl or substitutedethynyl designates substitution of the β position of vinyl or ethynyl bya halogen such as bromine, chlorine, fluorine or iodine, or substitutionby an alkyl of 1 to 5 carbon atoms such as methyl, ethyl, propyl, butyl,pentyl and the like, or aralkyl such as benzyl, p-chlorobenzyl,p-nitrobenzyl and the like, or aryl such as phenyl, p-nitrophenyl,p-tolyl, p-anisyl, naphtyl and the like; lower alkanoyl (acyl groups)containing 1 to 20 carbons such as formyl, acetyl, propionyl,isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl,caproyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl,stilligyl, palmitoyl, oleyl, linolenyl, arachidonyl and the like; loweraryl containing 1 to 20 carbons such as phenyl, p-tolyl, p-chlorophenyl,p-aminophenyl, p-nitrophenyl, p-anisyl and the like; lower aroylcontaining 1 to 20 carbons such as benzoyl and naphthoyl, where thearomatic group may be additionally substituted by alkyl, alkoxy, halo,or nitro moieties such as p-tolnoyl, p-anisoyl, p-chlorobenzoyl,p-nitrobenzoyl or 2,4-dinitrobenzoyl, pentafluorobenzoyl and the like,or another aroyl such as benzyloxybenzoyl and the like; lower aralkylcontaining 1 to 20 carbons such as benzyl, benzhydryl, p-chlorobenzyl,m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, pentaflourobenzyl andthe like; amino or alkylamino containing 1 to 20 carbons such as amonoalkyl- or monoaralkylamino groups like methylamino, ethylamino,propylamino or benzylamino and the like, dialkylamino such asdimethylamino, diethylamino, dibenzylamino, pyrrolidino, piperidino ormolpholino, and the like.

Thus, in certain embodiments, R¹ is hydrogen, hydroxyl, sulfyhydryl,amino, substituted amino, hydroxymethyl, monomethoxy, halogen,pseudohalogen, or a lower hydrocarbon (which hydrocarbon can besubstituted or unsubstituted) containing from 1 to 20 atoms. In aparticular embodiment, R¹ is a lower hydrocarbon selected from alkyl,substituted alkyl, alkenyl, alkanoyl, aryl, aroyl, aralkyl, oralkylamino. In a particular embodiment, R¹ is a lower hydrocarbonsubstituted with alkoxy, substituted alkoxy, imidate, arylthio, or(substituted aryl) thio. In other embodiments, R¹ is a lower alkylselected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl and pentyl. In other embodiments, R¹ is a lower alkenylselected from vinyl, substituted vinyl, ethynyl, or substituted ethynyl.In other embodiments, R¹ is a lower alkanoyl selected from formyl,acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl,valeryl, pivaloyl, caproyl, capryl, lauryl, myristyl, palmityl, stearyl,arachidyl, stilligyl, palmitoyl, oleyl, linolenyl, and arachidonyl. Inother embodiments, R¹ is lower aryl selected from phenyl, p-tolyl,p-chlorophenyl, p-aminophenyl, p-nitrophenyl, p-anisyl. In yet otherembodiments, R¹ is a lower aroyl selected from benzoyl and naphthoyl. Inother embodiments, R¹ is a lower aralkyl selected from benzyl,benzhydryl, p-chlorobenzyl, m-chlorobenzyl, p-nitrobenzyl,benzyloxybenzyl, or pentaflourobenzyl. In certain other embodiments, R¹is a lower alkylamino is selected from monoalkylamino, monoaralkylamino,dialkylamino, diaralkylamino, and benzylamino.

Compounds of interest include, but are not limited to, those of formula(I) where R¹ is selected from hydrogen, fluorine, trifluoromethyl,methyl, ethyl, propyl, butyl, isopropyl, isobutyl, acetyl, propionyl,butyryl, 2-bromovinyl, phenyl, benzyl, benzoyl, benzyloxybenzyl,benzylamino, alkyloxyalkyl, benzyloxyalkyl, imidatealkyl, arylthio, and(substituted aryl) thio. Thus, in certain embodiments, the compound isof formula (I), and R¹ is H, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH,(CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl,benzoyl, benzyloxybenzyl, benzyl-NH—, CH₃CH₂OCH₂, benzyl-O—CH₂, CH₃OCH₂,CH₃C(NH)—O—CH₂, or CH₃-phenyl-O—CH₂.

Examples of R² constituents of interest include, but are not limited to:hydrogen; hydroxyl; sulfyhydryl; halogen such as fluorine, chlorine,bromine or iodine, as well as pseudohalogen such as a loweralkylsulfonyl group of 1 to 5 carbons such as methyl-, ethyl-, propyl-,isopropyl-, butyl-, isobutyl-, tert-butyl-, and pentasulfonyl orarylsulfonyl such as benzene, p-toluene, p-nitrobenzenesulfonyl groups;lower alkyl containing 1 to 20 carbons such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl and the like, includingsubstituted lower alkyl such as aminomethyl, hydroxymethyl, methoxy,ethyloxy, propyloxy, and the like; lower alkenyl containing 1 to 20carbons such as vinyl and substituted vinyl, ethynyl and substitutedethynyl, where the substituted vinyl or substituted ethynyl designatessubstitution of the ß position of vinyl or ethynyl by a halogen such asbromine, chlorine, fluorine or iodine, or substitution by an alkyl of 1to 5 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, and thelike, or aralkyl such as benzyl, p-chlorobenzyl, p-nitrobenzyl, and thelike, or aryl such as phenyl, p-nitrophenyl, p-tolyl, p-anisyl, naphtyl,and the like; lower alkanoyl (acyl groups) and esters thereof of a mainchain containing 1 to 20 carbons such as formyl, acetyl, propionyl,isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl,caproyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl,stilligyl, palmitoyl, oleyl, linolenyl, arachidonyl, and the like; loweraryl containing 1 to 20 carbons such as phenyl, p-tolyl, p-chlorophenyl,p-aminophenyl, p-nitrophenyl, p-anisyl, and the like; lower aroylcontaining 1 to 20 carbons such as benzoyl and naphthoyl, where thearomatic group may be additionally substituted by alkyl, alkoxy, halo,or nitro moieties such as p-tolnoyl, p-anisoyl, p-chlorobenzoyl,p-nitrobenzoyl or 2,4-dinitrobenzoyl, pentafluorobenzoyl, and the like,or another aroyl such as benzyloxybenzoyl, and the like; lower aralkylcontaining 1 to 20 carbons such as benzyl, benzhydryl, p-chlorobenzyl,m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, pentaflourobenzyl, andthe like; lower aryloxy containing 1 to 20 carbons such as phenyloxy(i.e., O-phenyl), benzyloxy (i.e., O-benzyl), benzhydryloxy (i.e.,O-benzylhydryl), p-chlorobenzyloxy (i.e., O-(p-chlorobenzyl)),m-chlorobenzyloxy (i.e., O-(m-chlorobenzyl)), p-nitrobenzyloxy (i.e.,O-(p-nitrobenzyl)), (4-benzyloxybenzyl)-oxy (i.e., O-benzyloxybenzyl),or pentaflourobenzyloxy (i.e., O-pentaflourobenzyl); esters of aryloxys,such as lower aroyloxy (i.e., O-aroyl) containing 1 to 20 carbons suchas benzoyloxy (i.e., O-benzoyl), diphenylacetyloxy (i.e.,O-diphenylacetyl), p-chlorobenzoyloxy (i.e., O-(p-chlorobenzoyl)),m-chlorobenzoyloxy (i.e., O-(m-chlorobenzoyl)), p-nitrobenzoyloxy (i.e.,O-(p-nitrobenzoyl)), (4-benzyloxybenzoyl)-oxy (i.e.,O-benzyloxybenzoyl), or pentaflourobenzoyloxy (i.e.,O-pentaflourobenzoyl); amino or alkylamino containing 1 to 20 carbonssuch as a monoalkyl- or monoaralkylamino groups like methylamino,ethylamino, propylamino or benzylamino, and the like, dialkylamino suchas dimethylamino, diethylamino, dibenzylamino, pyrrolidino, piperidinoor molpholino, and the like.

Thus, in certain embodiments, R² is hydrogen, hydroxyl, sulfyhydryl,amino, hydroxymethyl, monomethoxy, halogen, pseudohalogen, or a lowerhydrocarbon (which hydrocarbon can be substituted or unsubstituted)containing from 1 to 20 atoms, and esters thereof. In a particularembodiment, R² is a lower hydrocarbon selected from alkyl, alkenyl,alkanoyl, aryl, aroyl, aryloxy, aroyloxy, aralkyl, or alkylamino. Inother embodiments, R² is a lower alkyl selected from methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl and pentyl. In otherembodiments, R² is a lower alkenyl selected from vinyl, substitutedvinyl, ethynyl, or substituted ethynyl. In other embodiments, R² is alower alkanoyl selected from formyl, acetyl, propionyl, isopropionyl,butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl, caproyl, capryl,lauryl, myristyl, palmityl, stearyl, arachidyl, stilligyl, palmitoyl,oleyl, linolenyl, and arachidonyl. In other embodiments, R² is loweraryl selected from phenyl, p-tolyl, p-chlorophenyl, p-aminophenyl,p-nitrophenyl, p-anisyl. In yet other embodiments, R² is a lower aroylselected from benzoyl and naphthoyl. In other embodiments, R² is a loweraralkyl selected from benzyl, benzhydryl, p-chlorobenzyl,m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, or pentaflourobenzyl. Inother embodiments, R² is a lower aryloxy selected from phenyloxy,benzyloxy, benzhydryloxy, p-chlorobenzyloxy, m-chlorobenzyloxy,p-nitrobenzyloxy, (4-benzyloxybenzyl)-oxy, or pentaflourobenzyloxy. Inother embodiments, R² is a lower aroyloxy selected from benzoyloxy,diphenylacetyloxy, p-chlorobenzoyloxy, m-chlorobenzoyloxy,p-nitrobenzoyloxy, (4-benzyloxybenzoyl)-oxy, or pentaflourobenzoyloxy.In certain other embodiments, R² is a lower alkylamino is selected frommonoalkylamino, monoaralkylamino, dialkylamino, and diaralkylamino.Thus, in certain embodiments, R² can not only be hydrogen or hydroxyl,but also an O-acyl, alkoxy, alkoxycarbonyl, alkoxycarbonylamino,O-alkyl, O-alkylene, O-alkynyl, O-aralkyl, O-aryl, O-aryloxy,O-carbohydrate, O-cycloalkenyl, O-cycloalkyl, O-heterocycloalkyl,O-heteroaryl. In addition, an S can substitute for the 0.

Compounds of interest include, but are not limited to, those of formula(I) where R² is selected from hydrogen, fluorine, trifluoromethyl,methyl, ethyl, propyl, butyl, isopropyl, isobutyl, acetyl, propionyl,butyryl, 2-bromovinyl, phenyl, phenyloxy, benzyl, benzoyl, benzoyloxyand benzyloxybenzyl. Thus, in certain embodiments, the compound is offormula (I), and R² is H, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH,(CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy,benzyl, benzoyl, benzoyloxy, or benzyloxybenzyl.

In specific embodiments of interest, the compound is of formula (I), andR² is hydrogen, hydroxyl, or an O-linked substituent. This includescompounds of formula (I), where R² is H, OH or C₆H₅C(O)O.

Examples of R³ of interest include, but are not limited to: hydrogen;hydroxyl; azido; sulfyhydryl; halogen; pseudohalogen; lower alkylcontaining 1 to 20 carbons such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl, and the like, including asubstituted lower alkyl such as aminomethyl, hydroxymethyl, methoxy,ethyloxy, propyloxy, and the like; lower alkanoyl (acyl) includingesters thereof of a main chain of 1 to 20 carbon atoms such as formyl,acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl,valeryl, pivaloyl, caproyl, capryl, lauryl, myristyl, palmityl, stearyl,arachidyl, stilligyl, palmitoyl, oleyl, linolenyl, arachidonyl, and thelike; lower aryl such as phenyl, p-nitrophenyl, p-tolyl, p-anisyl,naphtyl, and the like; lower aroyl (acyl radical of an aromatic acid) of1 to 20 carbons such as benzoyl and naphthoyl, where the aromatic groupmay be additionally substituted by alkyl, alkoxy, halo, or nitromoieties such as p-tolnoyl, p-anisoyl, p-chlorobenzoyl, p-nitrobenzoylor 2,4-dinitrobenzoyl, pentafluorobenzoyl, and the like; lower aryloxyof 1 to 20 carbons such as phenyloxy, benzyloxy, benzhydryloxy,p-chlorobenzyloxy, m-chlorobenzyloxy, p-nitrobenzyloxy,(4-benzyloxybenzyl)-oxy, or pentaflourobenzyloxy, and the like; as wellas esters of aryloxys, such as lower aroyloxy (O-aroyls) of 1 to 20carbons such as benzoyloxy, diphenylacetyloxy, p-chlorobenzoyloxy,m-chlorobenzoyloxy, p-nitrobenzoyloxy, (4-benzyloxybenzoyl)-oxy, orpentaflourobenzoyloxy, and the like. R³ may also be adamantoyl, orsubstituted adamantoyl.

Thus, in certain embodiments, R³ is hydrogen, hydroxyl, azido,sulfyhydryl, hydroxymethyl, halogen, or pseudohalogen. In otherembodiments, R³ is a lower hydrocarbon selected from alkyl, alkanoyl,aryl, aroyl, aryloxy, aroyloxy, or aralkyl. In other embodiments, R³ isa lower alkyl selected from methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl and pentyl. In other embodiments, R³ is a loweralkanoyl selected from formyl, acetyl, propionyl, isopropionyl, butyryl,isobutyryl, tert-butyryl, valeryl, pivaloyl, caproyl, capryl, lauryl,myristyl, palmityl, stearyl, arachidyl, stilligyl, palmitoyl, oleyl,linolenyl, and arachidonyl. In other embodiments, R³ is a lower arylselected from phenyl, p-tolyl, p-chlorophenyl, p-aminophenyl,p-nitrophenyl, p-anisyl, and the like. In other embodiments, R³ is alower aroyl selected from benzoyl and naphthoyl. In yet other certainembodiments, R³ is a lower aralkyl selected from benzyl, benzhydryl,p-chlorobenzyl, m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, orpentaflourobenzyl. In other embodiments, R³ is a lower aryloxy selectedfrom phenyloxy, benzyloxy, benzhydryloxy, p-chlorobenzyloxy,m-chlorobenzyloxy, p-nitrobenzyloxy, (4-benzyloxybenzyl)-oxy, orpentaflourobenzyloxy. In other embodiments, R³ is a lower aroyloxyselected from benzoyloxy, diphenylacetyloxy, p-chlorobenzoyloxy,m-chlorobenzoyloxy, p-nitrobenzoyloxy, (4-benzyloxybenzoyl)-oxy, orpentaflourobenzoyloxy. Thus, in certain embodiments, R³ can not only behydrogen or hydroxyl, but also an O-acyl, alkoxy, alkoxycarbonyl,alkoxycarbonylamino, O-alkyl, O-alkylene, O-alkynyl, O-aralkyl, O-aryl,O-aryloxy, O-carbohydrate, O-cycloalkenyl, O-cycloalkyl,O-heterocycloalkyl, O-heteroaryl. In addition, an S can substitute forthe O.

Compounds of interest are those of formula (I) where R³ is hydrogen,hydroxyl, halogen, azido, or an O-linked substituent. This includescompounds of formula (I) where R³ is selected from hydrogen, hydroxyl,n-butoxy, isobutyloxy, t-butyloxy, phenyloxy, benzyloxy, benzoyloxy, andpentafluorobenzoyloxy. Thus, in certain embodiments, the compound is offormula (I), and R³ is selected from H, OH, CH₃CH₂CH₂CH₂O,(CH₃)₂CH₂CH₂O, (CH₃)₃CO, C₆H50, benzoyloxy, and pentafluorobenzoyloxy.

In specific embodiments of interest, the compound is of formula (I),where R³ is H, OH, F, Cl, Br, I, N₃, or C₆H₅C(O)O. Of special interestis a compound of formula (I), where R³ is OH, or O-acyl (for example, anester such as C₆H₅C(O)O).

Examples of R⁴ include but are not limited to hydrogen; hydroxyl;sulfhydryl; halogen such as fluorine, chlorine, bromine or iodine; aminoor lower alkylamino. R⁴ also is exemplified by lower alkyl, with acylgroups which may be lower alkanoyl groups of 1 to 7 carbon atoms such asformyl, acetyl, propionyl, isopropionyl, butyryl, isobutyryl,tert-butyryl, and the like, and esters thereof. Thus, R⁴ can also bearoyl (and esters thereof such as O-linked aroyls, i.e., O-arolys orarolyoxy) such as benzoyl and naphthoyl wherein the aromatic group maybe additionally substituted by alkyl, alkoxy, halo, or nitro moietiessuch as p-tolnoyl, p-anisoyl, p-chlorobenzoyl, p-nitrobenzoyl or2,4-dinitrobenzoyl, and the like. Accordingly, in certain embodiments,R⁴ can not only be hydrogen or hydroxyl, but also an O-acyl, alkoxy,alkoxycarbonyl, alkoxycarbonylamino, O-alkyl, O-alkylene, O-alkynyl,O-aralkyl, O-aryl, O-aryloxy, O-carbohydrate, O-cycloalkenyl,O-cycloalkyl, O-heterocycloalkyl, O-heteroaryl. In addition, an S cansubstitute for the O.

Thus, in certain embodiments, R⁴ is hydrogen; hydroxyl; sulfhydryl;halogen, amino aminomethyl, or aminodimethyl. In other embodiments, R⁴is a lower alkyl, acyl, aroyl, or aroyloxy. This includes a specificembodiment, where the compound of formula (I) is one where R⁴ ishydrogen, flourine, hydroxyl, amino, aminomethyl, aminodimethyl,t-butyloxy, phenyloxy or benzoyloxy (for example, a compound of formula(I), where R⁴ is H, F, OH, NH₂, NHCH₃, N(CH₃)₂, (CH₃)₃CO, C₆H₅O orC₆H₅C(O)O).

Compounds of particular interest are those of formula (I) where R⁴ ishydrogen, hydroxyl, or an O-linked substituent. In specific embodiments,the compound is of formula (I), where R⁴ is H, OH or C₆H₅C(O)O. Ofspecial interest is a compound of formula (I), where R⁴ is OH, or O-acyl(for example, an ester such as C₆H₅C(O)O).

Other compounds of interest are compounds of formula (I) where: R¹ is H,F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂,CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R²is H, OH, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂,CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl, benzoyl,benzoyloxy, or benzyloxybenzyl, and where R³ and R⁴ are each hydroxyl.These include the compounds: 2,2′-anhydrouridine;2,2′-anhydro-5-fluorouridine; 2,2′-anhydro-5-trifluoromethyluridine;2,2′-anhydro-5-methyluridine; 2,2′-anhydro-5-ethyluridine;2,2′-anhydro-5-propyluridine; 2,2′-anhydro-5-isopropyluridine;2,2′-anhydro-5-isobutyluridine; 2,2′-anhydro-5-methylacyluridine;2,2′-anhydro-5-propylacyluridine; 2,2′-anhydro-5-(2-bromovinyl)-uridine;2,2′-anhydro-5-phenylluridine; 2,2′-anhydro-5-benzyluridine;2,2′-anhydro-5-benzyoluridine; and2,2′-anhydro-5-(benzyloxybenzyl)-uridine. Of special interest is2,2′-anhydro-5-methyluridine, or the pharmaceutically acceptable salts,solvates, hydrates, and prodrug forms thereof, and stereoisomersthereof.

Additional compounds of interest are compounds of formula (I) where: R¹is H, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂,CH₃(O)CCH₂,

CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R²is H, OH, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂,CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl,benzyloxy, benzoyl, benzoyloxy, or benzyloxybenzyl, and where R³ ishydroxyl, and R⁴ is benzoyloxy. These include the compounds:3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-fluorouridine;3′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;3′-O-benzoyl-2,2′-anhydro-5-ethyluridine;3′-O-benzoyl-2,2′-anhydro-5-propyluridine;3′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;3′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;3′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;3′-O-benzoyl-2,2′-anhydro-5-phenylluridine;3′-O-benzoyl-2,2′-anhydro-5-benzyluridine;3′-O-benzoyl-2,2′-anhydro-5-benzyoluridine; and3′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine. Of specificinterest is 3′-O-benzoyl-2,2′-anhydro-5-methyluridine, or thepharmaceutically acceptable salts, solvates, hydrates, and prodrug formsthereof, and stereoisomers thereof.

Also of interest are compounds of formula (I) where: R¹ is H, F, CF₃,CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂,CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R²is H, OH, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂,CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl,benzyloxy, benzoyl, benzoyloxy, or benzyloxybenzyl, and where R³ isbenzoyloxy, and R⁴ is hydroxyl. These include the compounds:5′-O-benzoyl-2,2′-anhydrouridine;5′-O-benzoyl-2,2′-anhydro-5-fluorouridine;5′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;5′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydro-5-ethyluridine;5′-O-benzoyl-2,2′-anhydro-5-propyluridine;5′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;5′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;5′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;5′-O-benzoyl-2,2′-anhydro-5-phenylluridine;5′-O-benzoyl-2,2′-anhydro-5-benzyluridine;5′-O-benzoyl-2,2′-anhydro-5-benzyoluridine; and5′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine. Of specificinterest is 5′-O-benzoyl-2,2′-anhydro-5-methyluridine, or thepharmaceutically acceptable salts, solvates, hydrates, and prodrug formsthereof, and stereoisomers thereof.

The 2,2′-anhydropyrimidine compounds of the invention may be incompositions that contain single stereoisomers, mixtures ofstereoisomers, as well various derivatives thereof that can occur asequilibrium mixtures of tautomers. For instance, 2,2′-anhydropyrimidinesaccording to formula (I) include four stereo centers with respect to thefurano ring, which includes the α and β anomers, and the L or D mirrorimage configurations. Examples of stereoisomers of the2,2′-anhydropyrimidine compounds of the invention are the β-D-isomer,β-L-isomer, α-D-isomer, and α-L-isomer, as well as tautomers andmixtures including α,β-D-isomers, α,β-L-isomers, α-DL-isomers, andβ-DL-isomers. Thus, in one embodiment, compositions are provided thatconsists essentially of a stereoisomer of a 2,2′-anhydropyrimidine thatis a β-D-isomer, β-L-isomer, α-D-isomer, or an α-L-isomer. Stereoisomersexhibiting improved activity on a molar basis or improved specificitywith respect to interfering with 5-FU efficacy are of special interest.

Stereoisomers of particular interest include:2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluorouracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyoluracil; and2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyoxybenzyl)uracil.Further stereoisomers of interest include:3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluororacil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;3′-O-benzoyl-2,2′-anhydro-1-(1-D-arabinofuranosyl)-5-isobutyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyoluracil; and3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyoxybenzyl)uracil.Additional stereoisomers of interest include:5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluorouracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyoluracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyoxybenzyl)uracil.

Examples of other analogs or derivatives of the 2,2′-anhydropyrimidinesof the invention, and stereoisomers thereof include:3′-O-acetyl-2,2′-anhydro-5-propyluridine(3′-O-acetyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propyluracil); and3′-O-acetyl-2,2′-anhydro-5-isopropyluridine(3′-O-acetyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil);as well as the 2,2′-anhydrocytidines, and analogs and derivativesthereof, of which the stereoisomer2,2′-anhydro-1-(β-D-arabinofuranosyl)cytosine is one example.

As noted above, stereoisomers and the various 2,2′-anhydropyrimidines ofparticular interest are those which exhibit improved activity on a molarbasis, or improved specificity with respect to not interfering with 5-FUefficacy. Such compounds can be readily selected for this purpose bycomparing against a matrix of compounds of particular interest, such asthose illustrated in Table 1 (where the compound is of formula (I)).

TABLE 1 The compound is of formula (I) Compound Stereoisomer R¹ R² R³ R⁴I-a β-D-isomer H H OH OH I-b β-D-isomer CH₃ H OH OH I-c β-D-isomerCH₃CH₂ H OH OH I-d β-D-isomer CH₃CH₂CH H OH OH I-e β-D-isomer BrCH═CH HOH OH I-f β-D-isomer C₆H₅CH₂ H OH OH I-g β-D-isomer H H C₆H₅C(O)O OH I-hβ-D-isomer CH₃ H C₆H₅C(O)O OH I-i β-D-isomer CH₃CH₂ H C₆H₅C(O)O OH I-jβ-D-isomer CH₃CH₂CH H C₆H₅C(O)O OH I-k β-D-isomer BrCH═CH H C₆H₅C(O)O OHI-l β-D-isomer C₆H₅CH₂ H C₆H₅C(O)O OH I-m β-D-isomer F—C₆H₅CH₂ H OH OHI-n β-D-isomer NO₂—C₆H₅CH₂ H OH OH I-o β-D-isomer NH₂—C₆H₅CH₂ H OH OHI-p β-D-isomer Cl—C₆H₅CH₂ H OH OH I-q β-D-isomer Alkyl- H OH OH C₆H₅CH₂I-r β-D-isomer Methoxy- H OH OH C₆H₅CH₂ I-s β-D-isomer Thiol- H OH OHC₆H₅CH₂ I-t β-D-isomer F—C₆H₅CH₂ H C₆H₅C(O)O OH I-u β-D-isomerNO₂—C₆H₅CH₂ H C₆H₅C(O)O OH I-v β-D-isomer NH₂—C₆H₅CH₂ H C₆H₅C(O)O OH I-wβ-D-isomer Cl—C₆H₅CH₂ H C₆H₅C(O)O OH I-x β-D-isomer Alkyl- H C₆H₅C(O)OOH C₆H₅CH₂ I-y β-D-isomer Methoxy- H C₆H₅C(O)O OH C₆H₅CH₂ I-z β-D-isomerThiol- H C₆H₅C(O)O OH C₆H₅CH₂ I-a′ β-D-isomer H OH H OH I-b′ β-D-isomerCH₃ OH H OH I-c′ β-D-isomer CH₃CH₂ OH H OH I-d′ β-D-isomer CH₃CH₂CH OH HOH I-e′ β-D-isomer BrCH═CH OH H OH I-f′ β-D-isomer C₆H₅CH₂ OH H OH I-g′β-D-isomer H C₆H₅C(O)O H OH I-h′ β-D-isomer CH₃ C₆H₅C(O)O H OH I-l′β-D-isomer CH₃CH₂ C₆H₅C(O)O H OH I-j′ β-D-isomer CH₃CH₂CH C₆H₅C(O)O H OHI-k′ β-D-isomer BrCH═CH C₆H₅C(O)O H OH I-l′ β-D-isomer C₆H₅CH₂ C₆H₅C(O)OH OH I-m′ β-D-isomer F—C₆H₅CH₂ OH H OH I-n′ β-D-isomer NO₂—C₆H₅CH₂ OH HOH I-o′ β-D-isomer NH₂—C₆H₅CH₂ OH H OH I-p β-D-isomer Cl—C₆H₅CH₂ OH H OHI-q′ β-D-isomer Alkyl- OH H OH C₆H₅CH₂ I-r′ β-D-isomer Methoxy- OH H OHC₆H₅CH₂ I-s′ β-D-isomer Thiol- OH H OH C₆H₅CH₂ I-t′ β-D-isomer F—C₆H₅CH₂C₆H₅C(O)O H OH I-u′ β-D-isomer NO₂—C₆H₅CH₂ C₆H₅C(O)O H OH I-v′β-D-isomer NH₂—C₆H₅CH₂ C₆H₅C(O)O H OH I-w′ β-D-isomer Cl—C₆H₅CH₂C₆H₅C(O)O H OH I-x′ β-D-isomer Alkyl- C₆H₅C(O)O H OH C₆H₅CH₂ I-y′β-D-isomer Methoxy- C₆H₅C(O)O H OH C₆H₅CH₂ I-z′ β-D-isomer Thiol-C₆H₅C(O)O H OH C₆H₅CH₂

As mentioned above, the compounds in Table I are illustrative but notlimiting. For example, R⁴ can be not only hydroxyl, but also an O-acyl,alkoxy, alkoxycarbonyl, alkoxycarbonylamino, O-alkyl, O-alkylene,O-alkynyl, O-aralkyl, O-aryl, O-aryloxy, O-carbohydrate, O-cycloalkenyl,O-cycloalkyl, O-heterocycloalkyl, O-heteroaryl. In addition, an S cansubstitute for the 0 and other combinations of the structural elementssuch as described herein, as well as other stereochemical orientations,are also possible.

In certain embodiments, acyl derivatives of the 2,2′-anyhydropyrimidinesof formula (I) are of interest. Thus, compounds of formula (I) includethose in which R¹,

R², R³ and R⁴ are as defined above, wherein at least one of R², R³ andR⁴ is an acyl derivative. By “acyl derivative” is intended a derivativeof a 2,2′-anyhydropyrimidine of formula (I) in which at least one of R²,R³ and R⁴ is a substantially nontoxic organic acyl substituentobtainable from a carboxylic acid that is attached to a hydroxyl groupon the ribose or pyrimidine ring of formula (I) through an esterlinkage.

Acyl derivatives of a 2,2′-anyhydropyrimidine compound of formula (I)include those in which R¹ is as defined above, and each R², R³ and R⁴ isindependently hydrogen, hydroxyl or an acyl radical, with the provisothat at least one of R², R³ and R⁴ is not hydrogen. In anotherembodiment, the acyl derivative of a 2,2′-anyhydropyrimidine is acompound of formula (I) in which R¹ and R² are as defined above, withthe proviso that R² is other than hydrogen, and each R³ and R⁴ isindependently hydroxyl or an acyl radical. In one embodiment, the acylderivative of a 2,2′-anyhydropyrimidine is a compound of formula (I) inwhich R¹ is as defined above, R² is hydrogen, and each R³ and R⁴ isindependently hydroxyl or an acyl radical. Of particular interest, is anacyl derivative of a 2,2′-anyhydropyrimidine compound of formula (I),wherein R¹ is methyl, R² is hydrogen, and each R³ and R⁴ isindependently hydroxyl or an acyl radical. Also of interest is an acylderivative of a 2,2′-anyhydropyrimidine compound of formula (I), whereinR¹ is methyl, R² is hydrogen, and each R³ and R⁴ is an acyl radical.

In general, the ester linkage(s) of an acyl derivative of formula (I)are cleavable under physiological conditions, either in vitro, such asin a cell-based system, and/or in vivo, such as through metabolism in abody. Thus, in certain embodiments, the acyl radical is a radical of ametabolite. Such acyl substituents include, but are not limited to,those derived from acetic acid, fatty acids, amino acids, lipoic acid,glycolic acid, lactic acid, enolpyruvic acid, pyruvic acid, orotic acid,acetoacetic acid, beta-hydroxybutyric acid, creatinic acid, succinicacid, fumaric acid, adipic acid, benzoic acid and p-aminobenzoic acid.Particular acyl substituents of interest are compounds which arenormally present in the body, either as dietary constituents or asintermediary metabolites, and which are essentially nontoxic whencleaved from the 2,2′-anyhydropyrimidine compound of interest in vivo.

Of particular interest are compositions comprising a3′-O-acyl-2,2′-anhydropyrimidine or derivative thereof. For example,acyl derivatives of interest are those that include a2,2′-anyhydropyrimidine compound of formula (I), where each R¹, R² andR³ is independently selected from selected from hydrogen, hydroxyl,sulfyhydryl, amino, hydroxymethyl, methoxy, halogen, pseudohalogen, anda substituted or unsubstituted lower hydrocarbon containing 1 to 20carbons, such as a lower hydrocarbon selected from alkyl, alkenyl,alkanoyl, aryl, aroyl, aralkyl and alkylamino, and esters thereof, andwhere R⁴ is an O-acyl radical.

In certain embodiments, the acyl derivatives include a2,2′-anyhydropyrimidine compound of formula (I), where R⁴ is an O-acylradical, and where the O-acyl radical comprises 1 to 10 carbon atoms,such as an O-acyl radical selected from aroyloxy, aralkoyloxy,heteroaroyloxy, and cycloalkoyloxy.

Accordingly, acyl derivatives of a 2,2′-anyhydropyrimidine compound offormula (I) include 3′-O-acyl-2,2′-anyhdropyrimidines,5′-O-acyl-2,2′-anyhdropyrimidines, 3′,5′-O-acyl-2,2′-anyhdropyrimidines,and derivatives thereof. For example, 3′-O-acyl-2,2′-anhydropyrimidinesor derivatives thereof include 3′-O-aroyl-2,2′-anhydropyrimidines, suchas a 3′-O-aroyl-2,2′-anhydrouridine or derivative thereof. An example ofparticular interest is 3′-O-benzoyl-2,2′-anhydrouridine or derivativethereof, such as 3′-O-benzoyl-2,2′-anhydro-5-methyluridine. Also ofinterest is a compound in which the3′-O-benzoyl-2,2′-anhydro-5-methyluridine is the stereoisomer3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil.

In some embodiments, acyl derivatives of a 2,2′-anyhydropyrimidinecompound of formula (I) include those where: R¹ is H, F, CF₃, CH₃,CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂,Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R² is H, OH, F,CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂,CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl, benzyloxy, benzoyl,benzyloxybenzyl, or acyl radical, and where each R³ and R⁴ isindependently hydroxyl or an acyl radical. These include the compounds:3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-fluorouridine;3′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;3′-O-benzoyl-2,2′-anhydro-5-ethyluridine;3′-O-benzoyl-2,2′-anhydro-5-propyluridine;3′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;3′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;3′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;3′-O-benzoyl-2,2′-anhydro-5-phenylluridine;3′-O-benzoyl-2,2′-anhydro-5-benzyluridine;3′-O-benzoyl-2,2′-anhydro-5-benzyoluridine; and3′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine;5′-O-benzoyl-2,2′-anhydrouridine;5′-O-benzoyl-2,2′-anhydro-5-fluorouridine;5′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;5′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydro-5-ethyluridine;5′-O-benzoyl-2,2′-anhydro-5-propyluridine;5′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;5′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;5′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;5′-O-benzoyl-2,2′-anhydro-5-phenylluridine;5′-O-benzoyl-2,2′-anhydro-5-benzyluridine;5′-O-benzoyl-2,2′-anhydro-5-benzyoluridine; and5′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine;3′,5′-O-benzoyl-2,2′-anhydrouridine;3′,5′-O-benzoyl-2,2′-anhydro-5-fluorouridine;3′,5′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-methyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-ethyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-propyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;3′,5′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;3′,5′-O-benzoyl-2,2′-anhydro-5-phenylluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-benzyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-benzyoluridine; and3′,5′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine; or thepharmaceutically acceptable salts, solvates, hydrates, and prodrug formsthereof, and stereoisomers thereof.

Of specific interest is 3′-O-benzoyl-2,2′-anhydro-5-methyluridine,5′-O-benzoyl-2,2′-anhydro-5-methyluridine, and3′,5′-O-benzoyl-2,2′-anhydro-5-methyluridine, or the pharmaceuticallyacceptable salts, solvates, hydrates, and prodrug forms thereof, andstereoisomers thereof. Of specific interest are the β-D-arabinofuranosylisomers of these compounds, or the pharmaceutically acceptable salts,solvates, hydrates, and prodrug forms thereof.

In another embodiment, compounds according to formula (I) of specificinterest are those where R¹ and R⁴ are as defined above, and R² and/orR³ is a cyclic hydrocarbyl. By “cyclic hydrocarbyl” is intended ahydrocarbon-based ring structure having from 3 to about 10 carbon atomsand having a single cyclic ring or multiple condensed rings that may besubstituted. Cyclic hydrocarbyls of interest are selected from aryl,aralkyl, aryloxy, aroyl, aroyloxy, heteroaryl, heteroaryloxy,heteroaroyloxy, cycloalkyl, cycloalkyloxy and cycloalkoyloxy. Thus,cyclic hydrocarbyls of special interest are O-linked to the ribose orpyrimidine ring of formula (I). Compounds where R² and/or R³ is a cyclichydrocarbyl exhibit improved activity on a molar basis, or improvedspecificity with respect to not interfering with 5-FU efficacy.

Accordingly, certain compounds of the invention comprise a 5′-O-(cyclichydrocarbyl)-2,2′-anhydropyrimidine or derivative thereof. Thisembodiment includes 5′-O-(cyclic hydrocarbyl)-2,2′-anhydro-5(R⁵)-uridineor derivatives thereof, where R⁵ is R¹ (e.g., R⁵═R¹ where “5(R⁵)” refersto and is the same as R¹ of formula (I)).

A compound of interest is 5′-O-aryl-2,2′-anhydropyrimidine or derivativethereof, of which various 2,2′-anhydrouridine derivatives are ofincluded. This includes compounds where the5′-O-aryl-2,2′-anhydropyrimidine is a 5′-O-aroyl-2,2′-anhydropyrimidine,such as: 5′-O-benzoyl-2,2′-anhydropyrimidine;5′-O-chlorobenzyl-2,2′-anhydropyrimidine;5′-O-nitrobenzyl-2,2′-anhydropyrimidine;5′-O-hydroxybenzyl-2,2′-anhydropyrimidine, and the like.

In one embodiment, compounds that exhibit improved activity on a molarbasis or improved specificity with respect to not interfering with 5-FUtherapy efficacy are the 5′-O-aryl-2,2′-anhydrouridines,5′-O-aroyl-2,2′-anhydrouridines, and derivatives thereof, such as5′-O-aryl-2,2′-anhydro-5(R⁴)-uridine,5′-O-aroyl-2,2′-anhydro-5(R⁴)-uridine, and their derivatives. Examplesinclude 5′-O-aryl-2,2′-anhydro-5-methyl-uridine;5′-O-aryl-2,2′-anhydro-5-ethyl-uridine;5′-O-aryl-2,2′-anhydro-5-propyl-uridine;5′-O-aryl-2,2′-anhydro-5-benzyl-uridine; and5′-O-aryl-2,2′-anhydro-5-(2-bromovinyl)-uridine; and derivativesthereof. Examples also include 5′-O-aroyl-2,2′-anhydro-5-methyl-uridine;5′-O-aroyl-2,2′-anhydro-5-ethyl-uridine;5′-O-aroyl-2,2′-anhydro-5-propyl-uridine;5′-O-aroyl-2,2′-anhydro-5-benzyl-uridine; and5′-O-aroyl-2,2′-anhydro-5-(2-bromovinyl)-uridine; and derivativesthereof. Compounds of specific interest include5′-O-benzoyl-2,2′-anhydro-5(R⁴)-uridines, such as5′-O-benzoyl-2,2′-anhydro-5-methyl-uridine;5′-O-benzoyl-2,2′-anhydro-5-ethyl-uridine;5′-O-benzoyl-2,2′-anhydro-5-propyl-uridine;5′-O-benzoyl-2,2′-anhydro-5-benzyl-uridine; and5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine.

Stereoisomers of interest include the 5′-O-(cyclichydrocarbyl)-2,2′-anhydropyrimidines which are the β-D-isomers. Examplesinclude, but are not limited to:5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluorouracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyoluracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyoxybenzyl)uracil.

As noted above, also of interest are analogues/derivatives of the abovecompounds, where such analogs/derivatives reduce 5-FU toxicity, suchthat 5-FU toxicity is reduced when the compounds are administered inconjunction with 5-FU therapy according to the subject invention. Asalso indicated above, an effective amount of uridine maximizing adjuvantis employed in the subject methods.

In certain embodiments, the amount of uridine maximizing adjuvantemployed is more than the amount of the 5-FU active agent employed. Incertain embodiments, the amount of uridine maximizing adjuvant is anamount that is less than equimolar to the amount of 5-FU active agentthat is administered. Typically, the amount of toxicity-reducingadjuvant that is administered is less than about 75%, less than about50%, less then about 25% and many embodiments less than about 15%, lessthan about 10% and even less than about 5% or 1% than the amount of the5-FU active agent. In one embodiment, the effective amount is about 1%to 50% of the amount of the 5-FU active agent, such as about 3% to 40%,and including about 5% to 30% of the amount of the 5-FU active agent. Inother embodiments, the effective amount is the same as the amount of theactive agent, and in certain embodiments the effective amount is anamount that is more than the amount of the 5-FU active agent. Effectiveamounts can readily be determined empirically using the data provided inthe Experimental section below.

The 2,2′-anhydropyrimidine and derivatives thereof described above arecommercially available or can be conventionally prepared by techniquesknown to one of skill in the art. For example, representative patentsdescribing various 2,2′-anhydropyrimidine and derivatives, includingintermediates and precursors, analysis, as well as thesynthesis/preparation thereof, include U.S. Pat. Nos. 3,975,367;4,145,531; 4,230,698; 4,247,544; 4,544,740; 4,604,382; 4,613,604;4,681,933; 4,841,039; 4,916,122; 4,987,224; 5,008,384; 5,077,280;5,084,445; 5,141,943; 5,190,926; 5,212,293; 5,278,167; 5,384,396;5,455,339; 5,476,855; 5,596,093; 5,610,292; 5,721,241; 5,723,449;5,739,314; 5,760,202; 5,889,013; 5,861,493; 6,060,592; 6,090,932;6,222,025; 6,369,040; 6,642,367; 6,670,461; 6,867,290; and 7,176,295;the disclosures of which are herein incorporated by reference. See also,the following references: Veres et al., Biochem Pharmacol. 34(10):1737(1985); Veres et al., Drugs Exp Clin Res. 13(10): 615 (1987); el Konuiet al, Mol. Pharmacology 34:104 (1988); Cienfuegos et al. Org. Lett.7(11):2161 (2005); Choi et al., Nucleosides Nucleotides Nucleic Acids22(5-8):547 (2003); Rodriquez et al., J Med Chem 37(20):3389 (1994);McGee, D. P. C. et al., “Novel Nucleosides via IntramolecularFunctionalization of 2,2′ Anahydrouridine Derivatives”, Tetr. Lett.,37(12):1995 (1996); Machulla et al. J. Nucl. Med. 42(5):257 (2001);Czernecki S. et al. Nucleosides & Nucleotides 14:1227 (1995);Heterocyclic Chemistry (3rd Edition), Thomas. L. Gilchrist, PrenticeHall (1997); Movassaghi, M. and M. D. Hill, J. Am. Chem. Soc.128(44):14254 (2006); Brown, D. J. Heterocyclic Compounds: ThePyrimidines. Vol 52. New York: lnterscience, 1994; Eaton, (1995) Annu.Rev. Biochem. 64, 837; Usman and Cedergreen TIBS 17:334 (1992); Greeneand Wuts (1991) Protective Groups in Organic Synthesis, 2nd Ed., Wileylnterscience); Moffatt, (1979) Nucleoside Analogues, Ed. Walker, NY,Plenum.; Townsend, (1988) Chemistry of Nucleosides and Nucleotides, NY,Plenum; and Sproat, et al., (1991) Oligonucleotides and Analogues: APractical Approach, ed. F. Eckstein, NY. Oxford Univ. Press).

Of particular interest are 2,2′-anhydropyrimidines and derivativesthereof that are inhibitors of uridine phosphorylase. Uridinephosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleosidephosphorylase family of enzymes which catalyzes the phosphorolyticcleavage of the C—N glycoside bond of uridine, with the formation ofribose 1-phosphate and uracil (Timofeev et al., Acta Crystallogr Sect FStruct Biol Cryst Commun., 63: 852-854 (2007).

The scope of the present invention includes prodrugs of the 5-FU activeagent and the uridine maximizing adjuvant. Such prodrugs are, ingeneral, functional derivatives of the compounds that are readilyconvertible in vivo into the required compounds. Thus, in the methods ofthe present invention, the term “administering” encompassesadministering the compound specifically disclosed or with a compoundwhich may not be specifically disclosed, but which converts to thespecified compound in vivo after administration to the subject in needthereof. Conventional procedures for the selection and preparation ofsuitable prodrug derivatives are described, e.g., in Wermuth, “DesigningProdrugs and Bioprecursors” in Wermuth, ed. The Practice of MedicinalChemistry, 2d Ed., pp. 561-586 (Academic Press 2003). Prodrugs includeesters that hydrolyze in vivo (eg, in the human body) to produce acompound described herein suitable for the present invention. Suitableester groups include, without limitation, those derived frompharmaceutically acceptable, aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety has no more than 6 carbon atoms. Illustrativeesters include formates, acetates, propionates, butyrates, acrylates,citrates, succinates, and ethylsuccinates.

In some embodiments, the toxicity reducing adjuvant is administered inconjunction with a plasma uridine level modulator. A plasma uridinelevel modulator is an agent that changes the plasma uridine level of asubject following administration to the subject. In some instances, theplasma uridine modulator enhances the plasma uridine level in thesubject. While the magnitude of any enhancement may vary, in someinstances the magnitude of enhancement is 2-fold or greater, such as5-fold or greater, 10-fold or greater, 15-fold or greater, 20-fold orgreater, 25-fold or greater, or 50-fold or greater. A variety ofdifferent types of plasma uridine enhancing agents may be employed.Plasma uridine enhancing agents include, but are not limited to, uridineand sources thereof, uridine precursors as sources thereof, and uridinedegradation inhibitors, such as Uridine phosphorylase (UPase)inhibitors, uridine secretion inhibiting compounds and uridine renaltransport competitors.

Uridine and sources thereof include, but are not limited to meatproducts, such as fish, pig and cow liver, and pancreas, and the like;fungi related products, such as brewer's yeast, beer, mushrooms, and thelike; vegetable products, such as sugarcane, tomatoes, oats, algae,broccoli, and the like; salts, such as uridine phosphates, acylateduridine, and the like. Uridine and sources thereof which may be employedin embodiments of the invention include, but are not limited to, thosedescribed in U.S. Pat. Nos. 9,579,337; 6,316,426; and 5,470,838; thedisclosures of which compounds are incorporated herein by reference.

Uridine precursors and sources thereof include, but are not limited to:prodrugs of uridine, such as triphenyluridine, orotic acid, and thelike; prodrugs of uridine 5′-monophosphate, such as mono- and di-alkylesters, acyloxyalkyl esters, alkoxycarbonylmethyl esters, substitutedethyl and propyl esters, amidomethyl esters, benzyl esters phenylesters, phosphonamidates, cyclophosphate esters, and the like; uridineprodrugs containing mono-, di- or tri-esters of uridine, such as mono-,di-, and triacetyl uridine, and the like; uridine prodrugs containingmono, di- or tri-phosphates of uridine, such as uridine monophosphate,uridine diphosphate, uridine triphosphate, and the like; uridinehomodimers and their esters, such as U-P-U, and the like; heterodimersof dideoxynucleoside compounds and uridine or uridine phosphorylaseinhibitors, such as AZT-P-U and AZT-P-BAU; etc. Uridine precursors andsources thereof which may be employed in embodiments of the inventioninclude, but are not limited to, those described in U.S. Pat. Nos.5,723,449 and 7,737,128; the disclosures of which compounds areincorporated herein by reference.

Uridine phosphorylase (UPase) inhibitors include, but are not limitedto: benzylacyclouridine, benzyloxyacylouridine,aminomethyl-benzylacylouridine,aminomethyl-benzyloxybenzylacyclouridine,hydroxymethyl-benzylacyclouridine, hydroxymethyl-benzyloxybenzylacyclouridine, and the like; derivatives of 5-benzylbarbiturate, such as5-benzyloxybenzyl barbiturate;5-benzyloxybenzyl-1-(1-hydroxy-2-ethoxy)methyl) barbiturate;5-benzyloxybenzylacetyl-1-(1-hydroxy-2-ethoxy) methyl) barbiturate;5-benzyloxybenzyl-1-(1,3-dihydroxy 2-propoxy)methyl barbiturate;5-benzyloxybenzyl-1-1-hydroxy, 3-amino-2-propoxy)methyl) barbiturate;

5-benzyloxybenzyl-1-(2-(3-carboxypropionyloxy)ethoxy) methyl)barbiturate; 5-benzyl-1-(1-hydroxy-2-ethoxy) methyl) barbiturate;5-methoxybenzylacetyl barbiturate;5-benzyl-1-(1,3-dihydroxy-2-propoxy)methyl) barbiturate;5-benzyl-1-(1-hydroxy, 3-amino-2-propoxy)methyl) barbiturate; and5-benzyl-1-(2-(3-carboxypropionyloxy)ethoxy) methyl) barbiturate, andthe like. Upase inhibitors which may be employed in embodiments of theinvention include, but are not limited to, those described in U.S. Pat.Nos. 5,723,449; 5,141,943; 5,077,280; and 4,613,604; the disclosures ofwhich compounds are incorporated herein by reference.

Uridine secretion inhibiting compounds include, but are not limited todrugs, such as dilazep, hexobendine. Uridine secretion inhibitingcompounds which may be employed in embodiments of the invention include,but are not limited to, those described in U.S. Pat. Nos. 6,989,376 and5,567,689; the disclosures of which compounds are incorporated herein byreference.

Uridine renal transport competitors include, but are not limited todrugs, such as L-uridine, L-2′,3′-dideoxyuridine,D-2′,3′-dideoxyuridine. Uridine renal transport competitors which may beemployed in embodiments of the invention include, but are not limitedto, those described in U.S. Pat. Nos. 6,989,376; 5,723,449 and5,567,689; the disclosures of which compounds are incorporated herein byreference.

Formulations

Also provided are pharmaceutical compositions containing the 5-FU activeagent and/or the uridine maximizing adjuvant employed in the subjectmethods. Accordingly, the 5-FU active agent and/or the uridinemaximizing adjuvant in pharmaceutical compositions, e.g., in the form ofa pharmaceutically acceptable salt, can be formulated for oral, topicalor parenteral administration for use in the subject methods, asdescribed above. In certain embodiments, e.g., where the compounds areadministered as separate formulations (such as in those embodimentswhere they are administered sequentially), separate or distinctpharmaceutical compositions, each containing a different active agent,are provided. In yet other embodiments, a single formulation thatincludes both of the 5-FU active agent and the uridine maximizingadjuvant (i.e., one composition that includes both active agents) isprovided.

By way of illustration, the 5-FU active agent and/or the uridinemaximizing adjuvant can be admixed with conventional pharmaceuticallyacceptable carriers and excipients (i.e., vehicles) and used in the formof aqueous solutions, tablets, capsules, elixirs, suspensions, syrups,wafers, and the like. Such pharmaceutical compositions contain, incertain embodiments, from about 0.1% to about 90% by weight of theactive compound, and more generally from about 1% to about 30% by weightof the active compound. The pharmaceutical compositions may containcommon carriers and excipients, such as corn starch or gelatin, lactose,dextrose, sucrose, microcrystalline cellulose, kaolin, mannitol,dicalcium phosphate, sodium chloride, and alginic acid. Disintegratorscommonly used in the formulations of this invention includecroscarmellose, microcrystalline cellulose, corn starch, sodium starchglycolate and alginic acid.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s), for example, ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, with a suspendingagent, preservative, surfactant, wetting agent, flavoring or coloringagent. Alternatively, a liquid formulation can be prepared from areconstitutable powder.

For example, a powder containing active compound, suspending agent,sucrose and a sweetener can be reconstituted with water to form asuspension; and a syrup can be prepared from a powder containing activeingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample, polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example, by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example, liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose. Lubricants that canbe used include magnesium stearate or other metallic stearates, stearicacid, silicone fluid, talc, waxes, oils, and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. Additionally, it may bedesirable to add a coloring agent to make the dosage form moreattractive in appearance or to help identify the product.

The compounds of the invention and their pharmaceutically acceptablesalts that are active when given parenterally can be formulated forintramuscular, intrathecal, or intravenous administration.

A typical composition for intramuscular or intrathecal administrationwill be of a suspension or solution of active ingredient in an oil, forexample, arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will be a sterile isotonicaqueous solution containing, for example, active ingredient and dextroseor sodium chloride, or a mixture of dextrose and sodium chloride. Otherexamples are lactated Ringer's injection, lactated Ringer's plusdextrose injection, Normosol-M and dextrose, Isolyte E, acylatedRinger's injection, and the like.

Optionally, a co-solvent, for example, polyethylene glycol, a chelatingagent, for example, ethylenediamine tetracetic acid, and an ant-oxidant,e.g., sodium metabisulphite may be included in the formulation.Alternatively, the solution can be freeze dried and then reconstitutedwith a suitable solvent just prior to administration. The compounds ofthe invention and their pharmaceutically acceptable salts which areactive on rectal administration can be formulated as suppositories. Atypical suppository formulation will generally consist of activeingredient with a binding and/or lubricating agent such as a gelatin orcocoa butter or other low melting vegetable or synthetic wax or fat.

The compounds of this invention and their pharmaceutically acceptablesalts which are active on topical administration can be formulated astransdermal compositions or transdermal delivery devices (“patches”).Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, herein incorporated by reference in itsentirety. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

In certain embodiments of interest, the 5-FU active agent and theuridine maximizing adjuvant are administered as a single pharmaceuticalformulation, that, in addition to including an effective amount of theactive agent and the toxicity-reducing adjuvant, includes other suitablecompounds and carriers, and may also be used in combination with otheractive agents. The present invention, therefore, also includespharmaceutical compositions comprising pharmaceutically acceptableexcipients. The pharmaceutically acceptable excipients include, forexample, any suitable vehicles, adjuvants, carriers or diluents, and arereadily available to the public. The pharmaceutical compositions of thepresent invention may further contain other active agents that are wellknown in the art.

One skilled in the art will appreciate that a variety of suitablemethods of administering a formulation of the present invention to asubject or host, e.g., patient, in need thereof, are available, and,although more than one route can be used to administer a particularformulation, a particular route can provide a more immediate and moreeffective reaction than another route. Pharmaceutically acceptableexcipients are also well-known to those who are skilled in the art andare readily available. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there are a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following methods and excipients are merelyexemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like, containing, in addition tothe active ingredient, such excipients as are known in the art.

The subject formulations of the present invention can be made intoaerosol formulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as for use in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Formulations suitable for topical administration may be presented ascreams, gels, pastes, or foams, containing, in addition to the activeingredient, and other such carriers that are known in the art to beappropriate.

Suppository formulations are also provided by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Suitable dosages for a given compound are readilydeterminable by those of skill in the art by a variety of means.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to cause a prophylactic ortherapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend on a varietyof factors including the strength of the particular compound employed,the condition of the animal, and the body weight of the animal, as wellas the severity of the illness and the stage of the disease. The size ofthe dose will also be determined by the existence, nature, and extent ofany adverse side-effects that might accompany the administration of aparticular compound. Suitable doses and dosage regimens can bedetermined by comparisons to anticancer or immunosuppressive agents thatare known to cause the desired growth inhibitory or immunosuppressiveresponse.

Optionally, the pharmaceutical composition may contain otherpharmaceutically acceptable components, such as buffers, surfactants,antioxidants, viscosity modifying agents, preservatives and the like.Each of these components is well-known in the art. For example, see U.S.Pat. No. 5,985,310, the disclosure of which is herein incorporated byreference.

Other components suitable for use in the formulations of the presentinvention can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). In anembodiment, the aqueous solution of cyclodextrin also contains dextrose,e.g., about 5% dextrose.

Utility

The subject methods find use in a variety of applications. In certainapplications, the methods are methods of modulating at least onecellular function, such as DHFR mediation of DNA synthesis and/orrepair. In this respect, the subject methods and compositions find usein known applications of 5-FU therapy, such as in treating diseases ordisorders that are capable of being treated using 5-FU. Use of thesubject compositions of the present invention is of particular utilityin, for example, the treatment of diseases and disorders including, butnot limited to, cancer, psoriasis, rheumatoid arthritis, Crohn'sdisease, and tissue-graft rejection, as well as in conditions requiringimmunosuppressive agents. In these capacities, use of the presentinventive compositions will result in reduced toxicity while retainingthe desired 5-FU therapy activity.

As such, the subject methods and compositions find use in therapeuticapplications in which 5-FU therapy is indicated. A representativetherapeutic application is in the treatment of cellular proliferativedisease conditions, e.g., cancers and related conditions, characterizedby abnormal cellular proliferation. Such disease conditions includecancer and neoplastic diseases and other diseases characterized by thepresence of unwanted cellular proliferation, e.g., hyperplasias, and thelike. Autoimmune diseases like multiple sclerosis also featureinappropriate proliferation of immune cells.

By treatment, is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g., a symptom associated with thecondition being treated or a side effect resulting from administrationof a drug. As such, treatment also includes situations where thepathological condition, or at least symptoms associated therewith, arecompletely inhibited, e.g., prevented from happening, or stopped, e.g.,terminated, such that the host no longer suffers from the condition, orat least the symptoms that characterize the condition.

A specific application of interest is the use of anhydro-nucleosides,particularly 2,2′-anhydropyrimidines and derivatives thereof, toameliorate 5-FU-induced mucositis. Thus, in certain embodiments, amethod is provided for the treatment of a host in need thereof aneffective amount of a 5-FU active agent in conjunction with an amount ofa uridine maximizing adjuvant effective to reduce 5-FU toxicity-inducedmucositis in the host, wherein the uridine maximizing adjuvant is a2,2′-anhydropyrimidine or derivative thereof. In a related embodiment,the 5-FU therapy-induced mucositis is stomatitis. In another relatedembodiment, the 5-FU therapy-induced mucositis is characterized by oneor more features selected from myelosuppression, weight loss,inflammation, and infection. Of specific interest is the use of2,2′-anhydro-5-methyluridine and acyl derivatives thereof as the uridinemaximizing adjuvant to reduce 5-FU therapy-induced mucositis in thehost.

Reduction of 5-FU therapy-induced mucositis is characterized by theprevention, mitigation, or reduction of the likelihood of onset ofmucositis resulting from treatment of a host with a 5-FU active agent.This includes treatment of a host in need thereof with an effectiveamount of a 5-FU active agent in conjunction with an amount of anuridine maximizing adjuvant effective to reduce 5-FU therapy-inducedmucositis in the host, where the uridine maximizing adjuvant improvesthe likelihood of successfully preventing or eliminating one or morefeatures of mucositis when it has occurred including: (i) prevention,that is, causing the clinical symptoms not to develop, e.g., preventingmyelosuppression, weight loss, inflammation, and/or infection, and/orpreventing progression of one or more of these features to a harmfulstate; (ii) inhibition, that is, arresting the development or furtherdevelopment of clinical symptoms, e.g., mitigating or completelyinhibiting an active (ongoing) feature of mucositis so that the featureis decreased to the degree that it is no longer seriously harmful, whichdecrease can include complete elimination of mucositis from the host;and/or (iii) relief, that is, causing the regression of clinicalsymptoms, e.g., causing a relief of myelosuppression, weight loss,inflammation, infection, and/or other symptoms caused by treatment ofthe host with an 5-FU active agent.

For example, mucositis severity, including oral mucositis (stomatitis),can easily be assessed by visual inspection of the mouth, throat and/oranal lesions associated with the condition, interrogation of testsubjects or patients (do you have soreness of the mouth or throat?) orby use of any, or all, three well accepted disease scales: thefive-grade World Health Organization (WHO) oral-toxicity scale (Miller AB et al., Cancer 1981; 47:207-214), the five-grade Radiation TherapyOncology Group (RTOG) acute radiation-morbidity scoring criteria formucous membranes, National Cancer Institute common toxicity criteria,version 2.0. Apr. 30, 1999, and the four-grade Western Consortium forCancer Nursing Research (WCCNR) revised staging system for oralmucositis (see, Assessing stomatitis: refinement of the WesternConsortium for Cancer Nursing Research (WCCNR) stomatitis staging system(Can Oncol Nurs J 1998; 8:160-165). Thus, the effect of treatment with auridine maximizing adjuvant can readily be determined using any, or all,of these test systems. In some instances, weight loss is diminished ascompared to a suitable control where the toxicity reducing agent is notemployed, such that is 80% or less, e.g., 70% or less, 60% or less, 50%or less, 40% or less, 30% or less, 25% or less, 20% or less, 15% orless, 10% or less, including 5% or less of the weight loss observed in asuitable control is observed with use the toxicity-reducing agent. Insome instances, no measurable weight loss occurs with thetoxicity-reducing agent is employed.

A variety of subjects are treatable according to the subject methods.Generally, such hosts are “mammals” or “mammalian,” where these termsare used broadly to describe organisms which are within the classMammalia, including the orders carnivore (e.g., dogs and cats), Rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the subjects will behumans.

In certain embodiments, the subjects will be subjects that have beendiagnosed for and are, therefore, in need of administration of theactive agent. In certain embodiments, the methods may include diagnosingthe subject for the presence of the disease condition to be treated byadministration of the active agent.

The subject methods find use in, among other applications, the treatmentof cellular proliferative disease conditions, including neoplasticdisease conditions, e.g., cancers, and autoimmune diseases. In suchapplications, an effective amount of the 5-FU active agent and uridinemaximizing adjuvant is administered to the subject in need thereof.Treatment is used broadly as defined above, to include at leastamelioration in one or more of the symptoms of the disease, as well as acomplete cessation thereof, as well as a reversal and/or completeremoval of the disease condition, i.e., a cure.

There are many disorders associated with a dysregulation of cellularproliferation, e.g., cellular proliferative disorders. The conditions ofinterest include, but are not limited to, conditions described below.

The subject methods may be employed in the treatment of a variety ofconditions where there is proliferation and/or migration of smoothmuscle cells, and/or inflammatory cells into the intimal layer of avessel, resulting in restricted blood flow through that vessel, e.g.,neointimal occlusive lesions. Occlusive vascular conditions of interestinclude atherosclerosis, graft coronary vascular disease aftertransplantation, vein graft stenosis, peri-anastomotic prosthetic graftstenosis, restenosis after angioplasty or stent placement, and the like.

Diseases where there is hyperproliferation and tissue re-modeling orrepair of reproductive tissue, e.g., uterine, testicular and ovariancarcinomas, endometriosis, squamous, and glandular epithelial carcinomasof the cervix, etc. are reduced in cell number by administration of thesubject compounds.

Tumors of interest for treatment include carcinomas, e.g., colon,duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic,renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma etc.; neurological malignancies, e.g.,neuroblastoma, gliomas, etc.; hematological malignancies, e.g.,childhood acute leukemia, acute myelogenous leukemias, acute lymphocyticleukemia, non-Hodgkin's lymphomas, chronic lymphocytic leukemia,malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-celllymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoidhyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, gestational choriocarcinoma, chorioadenoma destruens,hydatidiform mole, epidermoid cancers of the head and neck,trophoblastic neoplasms such as choriocarcinoma, chorioadenomadestruens, hydatidiform mole, etc., and the like.

Some cancers of particular interest include breast cancers, which areprimarily adenocarcinoma subtypes. Ductal carcinoma in situ (DCIS) isthe most common type of noninvasive breast cancer. In DCIS, themalignant cells have not metastasized through the walls of the ductsinto the fatty tissue of the breast. Infiltrating (or invasive) ductalcarcinoma (IDC) has metastasized through the wall of the duct andinvaded the fatty tissue of the breast. Infiltrating (or invasive)lobular carcinoma (ILC) is similar to IDC, in that it has the potentialmetastasize elsewhere in the body. About 10% to 15% of invasive breastcancers are invasive lobular carcinomas.

Also of interest is non-small cell lung carcinoma. Non-small cell lungcancer (NSCLC) is made up of three general subtypes of lung cancer.Epidermoid carcinoma (also called squamous cell carcinoma) usuallystarts in one of the larger bronchial tubes and grows relatively slowly.The size of these tumors can range from very small to quite large.Adenocarcinoma starts growing near the outside surface of the lung andmay vary in both size and growth rate. Some slowly growingadenocarcinomas are described as alveolar cell cancer. Large cellcarcinoma starts near the surface of the lung, grows rapidly, and thegrowth is usually fairly large when diagnosed. Other less common formsof lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignantmesothelioma.

Melanoma is a malignant tumor of melanocytes. Although most melanomasarise in the skin, they also may arise from mucosal surfaces or at othersites to which neural crest cells migrate. Melanoma occurs predominantlyin adults, and more than half of the cases arise in apparently normalareas of the skin. Prognosis is affected by clinical and histologicalfactors and by anatomic location of the lesion. Thickness and/or levelof invasion of the melanoma, mitotic index, tumor infiltratinglymphocytes, and ulceration or bleeding at the primary site affect theprognosis. Clinical staging is based on whether the tumor has spread toregional lymph nodes or distant sites. For disease clinically confinedto the primary site, the higher the chance of lymph node metastases andthe worse the prognosis is associated with greater thickness and depthof the local invasion of the melanoma. Melanoma can spread by localextension (through lymphatics) and/or by hematogenous routes to distantsites. Any organ may be involved by metastases, but lungs and liver arecommon sites.

Other proliferative diseases of interest relate to epidermalhyperproliferation, tissue remodeling and repair. For example, thechronic skin inflammation of psoriasis is associated with hyperplasticepidermal keratinocytes as well as infiltrating mononuclear cells,including CD4+ memory T cells, neutrophils and macrophages.

The methods of the present invention can provide a method of treatingmany, if not most, malignancies, including tumors derived from cellsselected from skin, connective tissue, adipose, breast, lung, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate,central nervous system (CNS), retina and blood, and the like.Representative cancers of interest include, but are not limited to,head, neck and lung tissue (e.g., head and neck squamous cell carcinoma,non-small cell lung carcinoma, small cell lung carcinoma)gastrointestinal tract and pancreas (e.g., gastric carcinoma, colorectaladenoma, colorectal carcinoma, pancreatic carcinoma); hepatic tissue(e.g., hepatocellular carcinoma), kidney and urinary tract (e.g.,dysplastic urothelium, bladder carcinoma, renal carcinoma, Wilms tumor),breast (e.g., breast carcinoma); neural tissue (e.g., retinoblastoma,oligodendroglioma, neuroblastoma, and malignant meningioma; skin (e.g.,normal epidermis, squamous cell carcinoma, basal cell carcinoma,melanoma, etc.).

The methods of the present invention also can provide a method oftreating hematological tissues (e.g., lymphoma, chronic myeloid leukemia(CML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), and the like.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to affect a prophylacticor therapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend on a varietyof factors, including the strength of the particular compound employed,the dose of the 5-FU active agent, the dosing regimen used for the 5-FUactive agent, the condition of the animal, and the body weight of theanimal, as well as the severity of the illness and the stage of thedisease.

The size of the dose will also be determined by the existence, nature,and extent of any adverse side-effects that might accompany theadministration of a particular compound.

In the treatment of some individuals with the compounds of the presentinvention, it may be desirable to use a high dose regimen in conjunctionwith a rescue agent for non-malignant cells. In such treatment, anyagent capable of rescue of non-malignant cells can also be employed,such as citrovorum factor, folate derivatives, or Leucovorin in additionto the adjuvant. Such rescue agents are well known to those of ordinaryskill in the art.

Applications in which the methods find use include, but are not limitedto, those described in U.S. Pat. Nos. 9,902,752; 9,012,444; 8,889,699;8,492,413; 8,440,398; 8,101,652; 7,910,580; 7,622,458; 7,345,039;7,026,301; 6,794,370; 6,670,335; 6,403,569; 5,843,917; 5,676,973;5,663,321; 5,610,160; 5,496,810; 5,457,187; 5,116,600; 5,089,503;5,077,055; 5,049,551; 5,047,521; 4,983,609; 4,914,105; 4,864,021;4,810,790; 4,719,213; 4,704,393; 4,652,570; 4,650,801; 4,631,342;4,622,325; 4,605,738; 4,558,12; 4,507,301; 4,497,815; 4,481,203;4,408,048; 4,394,505; 4,371,535; 4,349,552; 4,336,381; 4,328,229;4,256,885; 4,249,006; 4,248,999; 4,206,208; 4,196,202; 4,186,266;4,169,201; 4,130,648; 4,124,765; 4,122,251; 4,121,037; 4,113,949;4,110,537; 4,107,162; 4,088,646; 4,071,519; 4,032,524; 4,029,661;3,971,784; 3,960,864; 3,954,759; and 3,948,897; the disclosures of whichare herein incorporated by reference.

Kits & Systems

Also provided are kits and systems that find use in practicing thesubject methods, e.g., as described above. For example, kits and systemsfor practicing the subject methods may include one or morepharmaceutical formulations, which include one or both of the 5-FUactive agent and uridine maximizing adjuvant. As such, in certainembodiments the kits may include a single pharmaceutical composition,present as one, or more, unit dosages, where the composition includesboth the 5-FU active agent and uridine maximizing adjuvant. In yet otherembodiments, the kits may include two or more separate pharmaceuticalcompositions, each containing either a 5-FU active agent or a uridinemaximizing adjuvant.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits. For example, a kitaccording to one embodiment includes as a first component (a)instructions for using a uridine maximizing adjuvant, and as a secondcomponent (b) a pharmaceutical composition comprising a uridinemaximizing adjuvant, a 5-FU active agent, or a combination thereof.

Kits of specific interest are those that include a 2,2′-anhydropyrimidine pharmaceutical composition of the invention andsuitable for practicing the subject methods of the invention, such asfor reducing 5-FU active agent-induced mucositis, including stomatitis,and such as for treatment of a cellular proliferative disorder.

The term “system” as employed herein refers to a collection of a 5-FUactive agent and a uridine maximizing adjuvant, present in a single ordisparate composition, that are brought together for the purpose ofpracticing the subject methods. For example, separately obtained 5-FUactive agent and uridine maximizing adjuvant dosage forms broughttogether and co-administered to a subject, according to the presentinvention, are a system according to the present invention.

The following examples further illustrate the present invention butshould not be construed in any way as limiting its scope.

Experimental

I. Protection by the 2,2′-Anhydropyrimidine Test Articles Plus UridineProvided Better Protection Form 5-FU Induced Weight Loss than UridineAlone

The aim of this study was to explore whether uridine combined with auridine phosphorylase inhibitor provided better protection from weightloss produced by 5-FU compared to uridine alone. Summary results areprovided in FIG. 2. Weight loss is one of the cardinal features ofmucositis.

Four groups of female CD-1 mice were treated with daily ip injections ofeither PBS vehicle (Group 1), 80 mg/kg, 5-FU qdx5 (Groups 2), or 80mg/kg, 5-FU qdx5 combined with 750 mg/kg uridine, bid, or 80 mg/kg, 5-FUqdx5 combined with 750 mg/kg uridine and 120 mg/kg bid TK-90, arepresentative 2,2′-anhydropyrimidine test article, sandwiched beforeand after the uridine doses. Controls include PBS alone at timesmimicking the time for 5-FU and combined uridine and TK-90 treatments.There was n=6 animals in all groups.

Animals were sacrificed on Day 5 by cervical dislocation and properlydisposed. Animal weights were recorded daily for 5 days and the %difference between Day 0 and Day 5 at termination calculated.

Results from the study are provided as percent weight gain/loss frompre-dose weight on Day 0 in FIG. 2. Compared to the PBS vehicle control(blue), the 5-FU treatment (red) provided robust weight loss. By Day 5,the combined uridine and TK-90 treatment (green) provided robust,statistically significant protection compared to the 5-FU group alone(p<0.003) for Groups 2 and 4. The statistical difference between Groups2 and 3 was much less robust (p=0.036).

The combined treatments of uridine and a representative2,2′-anhydropyrimidine test article provided robust, statisticallysignificant protection much less than the protection compared to the5-FU alone group. The protection provided by uridine was statisticallysignificant but much less than the group with the representative2,2′-anhydropyrimidine test article.

Results from this experiment indicate that TK-90 is useful to mitigatemucositis associated with 5-FU, including when used in combination withsupplemental uridine or a supplement which, when administered, willprovide uridine. For certain situations, the supplemental uridine or auridine producing supplement is beneficial because a high uridineconcentration is useful to reverse, or protect from, the inducedtoxicity. Such situations include where there may be insufficientendogenous uridine present for TK-90 to protect uridine from clearanceand thereby elevate the protectant to an effective concentration. Aspreviously mentioned, the clearance of administered uridine is veryrapid, having a 3 minute or so half-life in man and animals. (Deng etal., “An adipo-biliary-uridine axis that regulates energy homeostasis,”Science (2017) 355(6330): eaaf5375.). As a result, the pharmacologicactivity of uridine itself without a clearance modulator can only havemarginal effects when administered at high doses, even doses that arehigh enough to cause significant toxicity.

II. Protection by the 2,2′-Anhydropyrimidine Test Articles Plus UridineProvided Better Protection Form 5-FU Induced Weight Loss than UridineAlone

The aim of this study was to confirm findings reported in FIG. 2 thaturidine combined with a uridine phosphorylase inhibitor provides betterprotection from a weight loss caused by 5-FU, compared to uridine alone.Summary results are provided in FIG. 3. Weight loss is one of thecardinal features of mucositis.

FIG. 3 depicts percent change in CD-1 male mouse weight for Day 5(termination) versus Day 0. Animals were dosed with either PBS ip for 5days; 400 mg/kg 5-FU, bid for one day, ip; or 400 mg/kg 5-FU, ip, bidfor one day plus 500 mg/kg, bid, ip, uridine for three days; or 400mg/kg 5-FU, ip, bid for one day plus 500 mg/kg, bid, ip, uridine forthree days and 100 mg/kg TK-90, ip, qid for three days. Four mice wereused for the PBS group all other groups were eight mice. Like the studysummarized in FIG. 2, uridine+TK-90 protected from the weight lossinduced by the 5-FU (p=0.001) better than uridine alone in this study(p=0.004) despite the much different experimental different design.

III. Protection by a 2,2′-Anhydropyrimidine Test Article Plus Uridinefrom a Decrease in Plasma Citrulline Caused by 5-FU

The chemotherapeutic challenge, 5-FU, was diminished plasma citrulline.The aim of this study was to explore whether a uridine phosphorylaseinhibitor plus uridine could prevent lowered citrulline induced by 5-FUbetter than uridine alone. Citrulline is a well-accepted surrogatemarker for mucositis.

FIG. 4 depicts citrulline concentrations measured in plasma obtained atsacrifice on Day 3 from C₅₇BL/6 female mice, approximately 20 g, treatedon Day 0 with 200 mg/kg 5-FU, ip. On Days 0-2, the uridine anduridine+TK-90 group animals were dosed with 250 mg uridine, bid. On Days0-2, the uridine+TK-90 group animals were treated with 700 mg/kg TK-90,bid, in addition to the 5-FU on Day 0 and the uridine on Days 0-2.Animals in the PBS group were treated on Day 0-2 with PBS, bid. Datafrom all six mice in the PBS, uridine and uridine plus TK-90 groups wereconsidered. Data from only five animals in the 5-FU group wereconsidered because results from one animal was considered an outlier(unusually low response from the injected 5-FU). Plasma citrulline is amarker for mucosal health. (Jones et al., “Citrulline as a Biomarker inthe Murine Total-Body Irradiation Model: Correlation of Circulating andTissue Citrulline to Small Intestine Epithelial Histopathology,” HealthPhys. (2015) 109(5):452-65.). Plasma citrulline is lowered withmucositis. (Fragkos et al., “Citrulline as a marker of intestinalfunction and absorption in clinical settings: A systematic review andmeta-analysis,” United European Gastroenterol J. (2018) 6(2):181-191.).TK-90 plus uridine reverses the decrease in plasma citrulline caused bythe 5-FU better than uridine alone.

Blood collections were done termination on Day 3 to animals anesthetizedwith ketamine (ip 100 mg/kg). Whole blood (˜0.8 mL) was drawn throughthe retro-orbital sinus using a heparin coated micro-hematocrit tube andcollected into a heparin microtainer tube. Blood samples weretransferred into fresh 1.5 mL microcentrifuge tubes and centrifuged for5 minutes at 10,000×g using an Eppendorf Minispin Plus stored in a 4° C.refrigerator. Following collection of the plasma samples, the animalswere sacrificed on Day 3 by cervical dislocation and properly disposed.The plasma samples were kept frozen until thawed for analysis.

Thawed plasma samples from the study were analyzed for citrulline usinga published LC/MS/MS-Isotope assay. (Jones et al., “Development andvalidation of a LC-MS/MS assay for quantitation of plasma citrulline forapplication to animal models of the acute radiation syndrome acrossmultiple species,” Anal Bioanal Chem. (2014) 406(19):4663-75.). Summaryresults provided as mean citrulline concentrations (μMolar) are providedin FIG. 4. Compared to the PBS vehicle control (blue), the 5-FUtreatment (red) provided a significant reduction in citrulline. Theuridine treatment (green) provided protection (p=0.036). However, theTK-90 treatment plus uridine (green) provided even better protection(p=0.019).

A representative 2,2′-anhydropyrimidine test article plus uridineprovided outstanding statistically significant protection from thelowered plasma citrulline, a widely accepted sign of mucositis, moreeffectively than uridine alone.

It is evident from the above results that the subject invention providesfor methods of improving the protection from the toxicity of 5-FUtherapy better than uridine alone. As such, the invention finds use in avariety of different applications and represents a significantcontribution to the art.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

1. A method of improving the therapeutic efficacy of uridine intreatment of a subject for a condition for which uridine treatment hasefficacy, the method comprising: administering to the subject effectiveamount of a 2,2′-anhydropyrimidine or derivative thereof in combinationwith a therapeutic amount of uridine or a uridine pro-drug.
 2. Themethod according to claim 1, wherein the 2,2′-anhydropyrimidine orderivative thereof is a compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof. wherein: each R¹, R²,R³ and R⁴ is independently selected from the group consisting ofhydrogen, substituted or unsubstituted heteroatom, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted aralkyl, carbohydrate, nucleic acid, amino acid,peptide, dye, fluorophore and polypeptide.
 3. The method according toclaim 2, wherein each R¹, R², R³ and R⁴ is independently selected fromthe group consisting of hydrogen, hydroxyl, sulfyhydryl, amino,hydroxymethyl, methoxy, halogen, pseudohalogen, and a substituted orunsubstituted lower hydrocarbon containing 1 to 20 carbons.
 4. Themethod according to claim 2, wherein the lower hydrocarbon is selectedfrom the group consisting of alkyl, alkenyl, alkanoyl, aryl, aroyl,aralkyl and alkylamino, and esters thereof.
 5. The method according toclaim 2, wherein R¹ is hydrogen, fluorine, methyl, ethyl, propyl,benzyl, or 2-bromovinyl; R² is hydrogen, hydroxyl fluorine, methyl,ethyl, propyl, benzyl, benzoyl, benzoyloxy, or 2-bromovinyl; and each R³and R⁴ is independently selected from the group consisting of hydroxyland benzoyloxy.
 6. The method according to claim 5, wherein R¹ ishydrogen or methyl; R² is hydrogen; and each R³ and R⁴ is independentlyselected from the group consisting of hydroxyl and benzoyloxy.
 7. Themethod according to claim 1, wherein the 2,2′-anhydropyrimidine orderivative thereof is selected from the group consisting of:2,2′-anhydro-5-methyluridine; 3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydrouridine; and5′-O-benzoyl-2,2′-anhydro-5-methyluridine.
 8. The method according toclaim 7, wherein the 2,2′-anhydropyrimidine or derivative thereof is2,2′-anhydro-5-methyluridine.
 9. The method according to claim 7,wherein the 2,2′-anhydropyrimidine or derivative thereof is3′-O-benzoyl-2,2′-anhydro-5-methyluridine.
 10. The method according toclaim 7, wherein the 2,2′-anhydropyrimidine or derivative thereof is5′-O-benzoyl-2,2′-anhydro-5-methyluridine.
 11. The method according toclaim 1, wherein the 2,2′-anhydropyrimidine or derivative thereofcomprises a stereoisomer.
 12. The method according to claim 11, whereinthe stereoisomer is selected from the group consisting of2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil. 13.The method according to claim 1, wherein the condition is 5-FUtherapy-induced toxicity. 14-31. (canceled)
 32. The method according toclaim 13, wherein the 5-FU therapy-induced toxicity is mucositis. 33.The method according to claim 32, wherein the mucositis is stomatitis.34. The method according to claim 13, wherein the amount of the2,2′-anhydropyrimidine or derivative thereof is administered inconjunction with a plasma uridine level modulator. 35-61. (canceled) 62.A method of treating a subject suffering from a cellular proliferativedisease condition, the method comprising: administering to the subjectan effective amount of a 5-FU active agent in conjunction with a uridinemaximizing adjuvant effective to reduce toxicity of the 5-FU activeagent so that the subject is treated for the cellular proliferativedisease condition, wherein the 5-FU toxicity reducing adjuvant is a2,2′-anhydropyrimidine, or derivative thereof.
 63. The method accordingto claim 62, wherein the 2,2′-anhydropyrimidine or derivative thereof isa compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof. wherein: each R¹, R²,R³, and R⁴ is independently selected from the group consisting ofhydrogen, substituted or unsubstituted heteroatom, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted aralkyl, carbohydrate, nucleic acid, amino acid,peptide, dye, fluorophore and polypeptide.
 64. The method according toclaim 63, wherein each R¹, R², R³ and R⁴ is independently selected fromselected from the group consisting of hydrogen, hydroxyl, sulfyhydryl,amino, hydroxymethyl, methoxy, halogen, pseudohalogen, and a substitutedor unsubstituted lower hydrocarbon containing 1 to 20 carbons.
 65. Themethod according to claim 64, wherein the lower hydrocarbon is selectedfrom the group consisting of alkyl, alkenyl, alkanoyl, aryl, aroyl,aralkyl and alkylamino, and esters thereof.
 66. The method according toclaim 63, wherein R¹ is hydrogen, fluorine, methyl, ethyl, propyl,benzyl, or 2-bromovinyl; R² is hydrogen, hydroxyl fluorine, methyl,ethyl, propyl, benzyl, benzoyl, benzoyloxy, or 2-bromovinyl; and each R³and R⁴ is independently selected from the group consisting of hydroxyland benzoyloxy. 67-99. (canceled)